Porcine mpl ligand

ABSTRACT

Isolated mpl ligand, isolated DNA encoding mpl ligand, and recombinant methods of preparing mpl ligand are disclosed. These mpl ligands are shown to influence the replication, differentiation or maturation of blood cells, especially megakaryocyte progenitor cells. Accordingly, these compounds are used for treatment of thrombocytopenia.

CROSS REFERENCE

This application is a continuation of earlier filed U.S. applicationSer. No. 08/176,553 filed Jan. 3, 1994 abandoned, which application isincorporated herein by reference and to which application priority isclaimed under 35 USC §120.

FIELD OF THE INVENTION

This invention relates to the isolation, purification andcharacterization of proteins that influence the replication,differentiation or maturation of blood cells, especially plateletprogenitor cells. This invention further relates to the cloning andexpression of a protein ligand capable of binding to and activating mpl,a member of the cytokine receptor superfamily. This application furtherrelates to the use of these proteins alone or in combination with othercytokines to treat thrombocytopenia.

BACKGROUND OF THE INVENTION

I. Megakaryocytopoiesis

It is known that bone marrow pluripotent stem cells differentiate intomegakaryocytic, erythrocytic, and myelocytic cell lines. It is believedthere is also a line of committed cells between stem cells andmegakaryocytes. The earliest recognizable member of the megakaryocyte(meg) family are the megakaryoblasts. These cells are initially 20 to 30μm in diameter having basophilic cytoplasm and a slightly irregularnucleus with loose, somewhat reticular chromatin and several nucleoli.Later, megakaryoblasts may contain up to 32 nuclei, but the cytoplasmremains sparse and immature. As maturation proceeds, the nucleus becomesmore lobulate and pyknotic, the cytoplasm increases in quantity andbecomes more acidophilic and granular. The most mature cells of thisfamily may give the appearance of releasing platelets at theirperiphery. Normally, less than 10% of megakaryocytes are in the blaststage and more than 50% are mature. Arbitrary morphologicclassifications commonly applied to the megakaryocyte series: aremegakaryoblast for the earliest form; promegakaryocyte or basophilicmegakaryocyte for the intermediate form; and mature (acidophilic,granular, or platelet-producing) megakaryocyte for the late forms. Themature megakaryocyte extends filaments of cytoplasm into sinusoidalspaces where they detach and fragment into individual platelets(Williams et al., Hematology, 1972).

Megakaryocytopoiesis is believed to involve several regulatory factors(Williams et al., Br. J. Haematol., 52:173 [1982] and Williams et al.,J. Cell Physiol. 110:101 [1982]). The early level ofmegakaryocytopoiesis is postulated as being mitotic, concerned with cellproliferation and colony initiation from CFU-meg but is not affected byplatelet count (Burstein et al., J. Cell Physiol. 109:333 [1981] andKimura et al., Exp. Hematol. 13:1048 [1985]). The later stage ofmaturation is non-mitotic, involved with nuclear polyploidization andcytoplasmic maturation and is probably regulated in a feedback mechanismby peripheral platelet number (Odell et al., Blood 48:765 [1976] andEbbe et al., Blood 32:787 [1968]). The existence of a distinct andspecific megakaryocyte colony-stimulating factor (meg-CSF) has beendisputed (Mazur, E., Exp. Hematol. 15:340-350 [1987]). Although.meg-CSF's have been partly purified from experimentally producedthrombocytopenia (Hill et al., Exp. Hematol. 14:752 [1986]) and humanembryonic kidney conditioned medium [CM] (McDonald et al., J. Lab. Clin.Med. 85:59 [1975]) and in man from aplastic anemia and idiopathicthrombocytopenic purpura urinary extracts (Kawakita et al., Blood 6:556[1983]) and plasma (Hoffman et al., J. Clin. Invest. 75:1174 [1985]),their physiological function is as yet unknown in most cases. Theconditioned medium of pokeweed mitogen-activated spleen cells (PWM-SpCM)and the murine myelomonocyte cell line WEHI-3 (WEHI-3CM) have been usedas megakaryocyte potentiators. PWM-SpCM contains factors enhancingCFU-meg growth (Metcalf et al., Pro. Natl. Acad. Sci., USA 72:1744-1748[1975]; Quesenberry et al., Blood 65:214 [1985]; and Iscove, N. N., inHematopoietic Cell Differentiation, ICN-UCLA Symposia on Molecular andCellular Biology, Vol. 10, Golde et al., eds. [New York, Academy Press]pp 37-52 [1978]), one of which is interleukin-3 (IL-3), a multilineagecolony stimulating factor (multi-CSF [Burstein, S. A., Blood Cells11:469 [1986]). The other factors in this medium have not yet beenidentified and isolated. WEHI-3 is a murine myelomonocytic cell linesecreting relatively large amounts of IL-3 and smaller amounts ofGM-CSF. IL-3 has been recently purified and cloned (Ihle et al., J.Immunol. 129:2431 [1982]) and has been found to potentiate the growth ofa wide range of hemopoietic cells (Ihle et al., J. Immunol. 13:282[1983]). IL-3 has also been found to synergize with many of the knownhemopoietic hormones or growth factors (Bartelmez et al., J. CellPhysiol. 122:362-369 [1985] and Warren et al., Cell 46:667-674 [1988]),including both erythropoietin (EPO) and H-1 (later known asinterleukin-1 or IL-1), in the induction of very early multipotentialprecursors and the formation of very large mixed hemopoietic colonies.

Other sources of megakaryocyte potentiators have been found in theconditioned media of murine lung, bone, macrophage cell lines,peritoneal exudate cells and human embryonic kidney cells. Despitecertain conflicting data (Mazur, E., Exp. Hematol. 15:340-350 [1987]),there is some evidence (Geissler et al., Br. J. Haematol. 60:233-238[1985]) that activated T lymphocytes rather than monocytes play anenhancing role in megakaryocytopoiesis. These findings suggest thatactivated T-lymphocyte secretions such as interleukins may be regulatoryfactors in meg development (Geissler et al., Exp. Hematol. 15:845-853[1987]). A number of studies on megakaryocytopoiesis with purified EPO(Vainchenker et al., Blood 54:940 [1979]; McLeod et al., Nature261:492-4 [1976]; and Williams et al., Exp. Hematol. 12:734 [1984])indicate that this hormone has an enhancing effect on meg colonyformation. More recently this has been demonstrated in both serum-freeand serum-containing cultures and in the absence of accessory cells(Williams et al., Exp. Hematol. 12:734 [1984]). EPO was postulated to beinvolved more in the single and two-cell stage aspects ofmegakaryocytopoiesis as opposed to the effect of PWM-SpCM which wasinvolved in the four-cell stage of megakaryocyte development. Theinteraction of all these factors on both early and late phases ofmegakaryocyte development remains to be elucidated.

Other documents of interest include: Eppstein et al., U.S. Pat. No.4,962,091; Chong, U.S. Pat. No. 4,879,111; Fernandes et al., U.S. Pat.No. 4,604,377; Wissler et al., U.S. Pat. No. 4,512,971; Gottlieb, U.S.Pat. No. 4,468,379; Kogan et al., U.S. Pat. No. 5,250,732; Kimura etal., Eur. J. Immunol., 20(9): 1927-1931 (1990); Secor, W. E. et al., J.of Immunol., 144(4): 1484-1489 (1990); Warren, D. J., et al., J. ofImmunol. 140(1): 94-99 (1988); Warren, M. K. et al., Exp. Hematol.,17(11): 1095-1099 (1989); Bruno, E., et al., Exp. Hematol., 17(10):1038-1043 (1989); Tanikawa et al., Exp. Hematol., 17(8): 883-888 (1989);Koike et al., Blood, 75(12): 2286-2291 (1990); Lotem, et al., Blood,75(5): 1545-1551 (1989); Rennick, D., et al., Blood, 73(7): 1828-1835(1989); and Clutterbuck, E. J., et al., Blood, 73(6): 1504-1512 (1989).

II. Thrombocytopenia

Platelets are critical elements of the blood clotting mechanism.Depletion of the circulating level of platelets, calledthrombocytopenia, occurs in various clinical conditions and disorders.Thrombocytopenia is commonly defined as a platelet count below 150×10⁹per liter. The major causes of thrombocytopenia can be broadly dividedinto three categories on the basis of platelet life span, namely; (1)impaired production of platelets by the bone marrow, (2) plateletsequestration in the spleen (splenomegaly), or (3) increased destructionof platelets in the peripheral circulation (e.g. autoimmunethrombocytopenia or chemo- and radiation therapy). Additionally, inpatients receiving large volumes of rapidly administered platelet-poorblood products, thrombocytopenia may develop due to dilution.

The clinical bleeding manifestations of thrombocytopenia depend on theseverity of thrombocytopenia, its cause, and possible associatedcoagulation defects. In general, patients with platelet counts between20 and 100×10⁹ per liter are at risk of excessive posttraumaticbleeding, while those with platelet counts below 20×10⁹ per liter maybleed spontaneously. These latter patients are candidates for platelettransfusion with attendant immune and viral risk. For any given degreeof thrombocytopenia, bleeding tends to be more severe when the cause isdecreased production rather than increased destruction of platelets; inthe latter situation, accelerated platelet turnover results in thecirculation of younger, larger and hemostatically more effectiveplatelets. Thrombocytopenia may result from a variety of disordersbriefly described below. A more detailed description may be found inSchafner, A. I., Thrombocytopenia and Disorders of Platelet Function,Internal Medicine, 3rd Ed., John J. Hutton et al., Eds., Little Brownand Co., Boston/Toronto/London, (1990).

(a) Thrombocytopenia Due to Impaired Platelet Production

Causes of congenital thrombocytopenia include constitutional aplasticanemia (Fanconi syndrome) and congenital amegakaryocyticthrombocytopenia, which may be associated with skeletal malformations.Acquired disorders of platelet production are caused by eitherhypoplasia of megakaryocytes or ineffective thrombopoiesis.Megakaryocytic hypoplasia can result from a variety of conditions,including marrow aplasia (including idiopathic forms or myelosuppressionby chemotherapeutic agents or radiation therapy), myelfibrosis,leukemia, and invasion of the bone marrow by metastatic tumor orgranulomas. In some situations, toxins, infectious agents, or drugs mayinterfere with thrombopoiesis relatively selectively; examples includetransient thrombocytopenias caused by alcohol and certain viralinfections and mild thrombocytopenia associated with the administrationof thiazide diuretics. Finally, ineffective thrombopoiesis secondary tomegaloblastic processes (folate or B₁₂ deficiency) can also causethrombocytopenia, usually with coexisting anemia and leukopenia.

Current treatment of thrombocytopenias due to decreased plateletproduction depends on identification and reversal of the underlyingcause of the bone marrow failure. Platelet transfusions are usuallyreserved for patients with serious bleeding complications or forcoverage during surgical procedures, since isoimmunization may lead torefractoriness to further platelet transfusions. Mucosal bleedingresulting from severe thrombocytopenia may be ameliorated by the oral orintravenous administration of the antifibrinolytic agents. Thromboticcomplications may develop, however, if antifibrinolytic agents are usedin patients with disseminated intravascular coagulation (DIC).

(b) Thrombocytopenia Due to Splenic Sequestration

Splenomegaly due to any cause may be associated with mild to moderatethrombocytopenia. This is a largely passive process (hypersplenism) ofsplenic platelet sequestration, in contrast to the active destruction ofplatelets by the spleen in cases of immunomediated thrombocytopeniadiscussed below. Although the most common cause of hypersplenism iscongestive splenomegaly from portal hypertension due to alcoholiccirrhosis, other forms of congestive, infiltrative, orlymphoproliferative splenomegaly are also associated withthrombocytopenia. Platelet counts generally do not fall below 50×10⁹ perliter as a result of hypersplenism alone.

(c) Thrombocytopenia Due to Nonimmune-mediated Platelet Destruction

Thrombocytopenia can result from the accelerated destruction ofplatelets by various nonimmunologic processes. Disorders of this typeinclude disseminated intravascular coagulation, prosthetic intravasculardevices, extracorporeal circulation of the blood, and thromboticmicroangiopathies such as thrombotic thrombocytic purpura. In all ofthese situations, circulating platelets that are exposed to eitherartificial surfaces or abnormal vascular intima either are consumed atthese sites or are damaged and then prematurely cleared by thereticuloendothelial system. Disease states or disorders in whichdisseminated intravascular coagulation (DIC) may arise are set forth ingreater detail in Braunwald et al: (eds), Harrison's Principles ofInternal Medicine, 11th Ed., p.1478, McGraw Hill (1987). Intravascularprosthetic devices, including cardiac valves and intra-aortic balloonscan cause a mild to moderate destructive thrombocytopenia and transientthrombocytopenia in patients undergoing cardiopulmonary bypass orhemodialysis may result from consumption or damage of platelets in theextracorporeal circuit.

(d) Drug-induced Immune Thrombocytopenia

More than 100 drugs have been implicated in immunologically mediatedthrombocytopenia. However, only quinidine, quinine, gold, sulfonamides,cephalothin, and heparin have been well characterized. Drug-inducedthrombocytopenia is frequently very severe and typically occursprecipitously within days while patients are taking the sensitizingmedication.

(e) Immune (Autoimmune) Thrombocytopenic Purpura (ITP)

ITP in adults is a chronic disease characterized by autoimmune plateletdestruction. The autoantibody is usually IgG although otherimmunoglobulins have also been reported. Although the autoantibody ofITP has been found to be associated with platelet membraneGPII_(b)III_(a), the platelet antigen specificity has not beenidentified in most cases. Extravascular destruction of sensitizedplatelets occurs in the reticuloendothelial system of the spleen andliver. Although over one-half of all cases of ITP are idiopathic, manypatients have underlying rheumatic or autoimmune diseases (e.g. systemiclupus erythematosus) or lymphoproliferative disorders (e.g. chroniclymphocytic leukemia).

(f) HIV-Induced ITP

ITP is an increasingly common complication of HIV infection (Morris etal., Ann. Intern. Med., 96: 714-717 [1982]), and can occur at any stageof the disease progression, both in patients diagnosed with the AcquiredImmune Deficiency Syndrome (AIDS), those with AIDS-related complex, andthose with HIV infection but without AIDS symptoms. HIV infection is atransmissible disease ultimately characterized by a profound deficiencyof cellular immune function as well as the occurrence of opportunisticinfection and malignancy. The primary immunologic abnormality resultingfrom infection by HIV is the progressive depletion and functionalimpairment of T lymphocytes expressing the CD4 cell surface glycoprotein(H. Lane et al., Ann. Rev. Immunol., 3:477 [1985]). The loss of CD4helper/inducer T cell function probably underlies the profound defectsin cellular and humoral immunity leading to the opportunistic infectionsand malignancies characteristic of AIDS (H. Lane supra).

Although the mechanism of HIV-associated ITP is unknown, it is believedto be different from the mechanism of ITP not associated with HIVinfection. (Walsh et al., N. Eng. J. Med., 311: 635-639 [1984]; andRatner, L., Am. J. Med., 86: 194-1981 [1989]).

III. Therapy

The therapeutic approach to the treatment of patients with HlV-inducedITP is dictated by the severity and urgency of the clinical situation.The treatment is similar for HIV-associated and non-HIV-related ITP, andalthough a number of different therapeutic approaches have been used,the therapy remains controversial.

Platelet counts in patients diagnosed with ITP have been successfullyincreased by glucocorticoid (e.g. prednisolone) therapy, however in mostpatients the response is incomplete, or relapse occurs when theglucocorticoid dose is reduced or its administration is discontinued.Based upon studies with patients having HIV-associated ITP, someinvestigators have suggested that glucocorticoid therapy may result inpredisposition to AIDS. Glucocorticoids are usually administered ifplatelet count falls below 20×10⁹/liter or when spontaneous bleedingoccurs.

For patients refractory to glucocorticoids, the compound4-(2-chlorphenyl)-9-methyl-2-[3-(4-morpholinyl)-3-propanon-1-yl]6H-thieno[3,2,f][1,2,4]triazolo[4,3,a,][1,4]diazepin (WEB 2086) has been successfully used to treat a severe case ofnon HIV-associated ITP. A patient having platelet counts of37,000-58,000/μl was treated with WEB 2086 and after 1-2 weeks treatmentplatelet counts increased to 140,000-190,000/μl. (EP 0361077A2 andLohman, H., et al., Lancet:1147 [1988]).

Although the optimal treatment for acquired amegacaryocyticthrombocytopenia purpura (AATP) is uncertain, antithymocyte globulin(ATG), a horse antiserum to human thymus tissue, has been shown toproduce prolonged complete remission (Trimble, M. S., et al., Am. J.Hematol.,37: 126-127 [1991]). A recent report however, indicates thatthe hematopoietic effects of ATG are attributable to thimerosal, wherepresumably the protein acts as a mercury carrier (Panella, T. J., andHuang, A. T., Cancer Research:50: 4429-4435 [1990]).

Good results have been reported with splenectomy. Splenectomy removesthe major site of platelet destruction and a major source ofautoantibody production in many patients. This procedure results inprolonged treatment-free remissions in a large number of patients.However, since surgical procedures are generally to be avoided in immunecompromised patients, splenectomy is recommended only in severe cases ofHIV-associated ITP, in patients who fail to respond to 2 to 3 weeks ofglucocorticoid treatment, or do not achieve sustained response afterdiscontinuation of glucocorticoid administration. Based upon currentscientific knowledge, it is unclear whether splenectomy predisposespatients to AIDS.

In addition to prednisolone therapy and splenectomy, certain cytotoxicagents, e.g. vincristine, and azidothimidine (AZT, zidovudine) also showpromise in treating HIV-induced ITP however, the results arepreliminary.

It will be appreciated from the foregoing that one way to treatthrombocytopenia would be to obtain an agent capable of accelerating thedifferentiation and maturation of megakaryocytes or precursors thereofinto the platelet-producing form. Considerable efforts have beenexpended on identifying such an agent, commonly referred to as“thrombopoeitin” (TPO). Thrombopoeitin activity was observed as early as1959 (Rak et al., Med. Exp.1:125) and attempts to characterize andpurify this agent have continued to the present day. While reports ofpartial purification of thrombopoeitin-active polypeptides exist (see,for example, Tayrien et al., J. Biol. Chem. 262:3262 [1987] and Hoffmanet al., J. Clin. Invest. 75:1174 [1985]), others have postulated thatthrombopoeitin is not a discrete entity in its own right but rather issimply the polyfunctional manifestation of a known hormone (IL-3,Sparrow et al., Prog. Clin. Biol. Res.,215:123 [1986]). Regardless ofits form or origin, a molecule possessing thrombopoetic activity wouldbe of significant therapeutic value. Although no protein has beenunambiguously identified as thrombopoeitin, considerable interestsurrounds the recent discovery that mpl, a putative cytokine receptor,may transduce a thrombopoietic signal.

IV. Mpl is a Cytokine Receptor

It is believed that the proliferation and maturation of hematopoieticcells is tightly regulated by factors that positively or negativelymodulate pluripotential stem cell proliferation and multilineagedifferentiation. These effects are mediated through the high-affinitybinding of extracellular protein factors to specific cell surfacereceptors. These cell surface receptors share considerable homology andare generally classified as members of the cytokine receptorsuperfamily. Members of the superfamily include receptors for: IL-2(beta and gamma chains) (Hatakeyama et al., Science 244:551-556 [1989];Takeshita et al., Science 257:379-382 [1991]), IL-3 (Itoh et al.,Science 247:324-328 [1990]; Gorman et al., Proc. Natl. Acad. Sci. USA87:5459-5463 [1990]; Kitamura et al., Cell 66:1165-1174 [1991a];Kitamura et al. Proc. Natl. Acad. Sci. USA 88:5082-5086 [1991b]), IL-4(Mosley et al., Cell 59:335-348 [1989], IL-5 (Takaki et al. EMBO J.9:4367-4374 [1990]; Tavernier et al., Cell 66:1175-1184 [1991]), IL-6(Yamasaki et al., Science 241:825-828 [1988]; Hibi et al. Cell63:1149-1157 [1990]), IL-7 (Goodwin et al. Cell 60:941-951 [1990]), IL-9(Renault et al. Proc. Natl. Acad. Sci. USA 89:5690-5694 [1992]),granulocyte-macrophage colony-stimulating factor (GM-CSF) (Gearing etal., EMBO J. 8:3667-3676 [1991]; Hayashida et al. Proc. Natl. Acad. Sci.USA 244:9655-9659 [1990]), granulocyte colony-stimulating factor (G-CSF)(Fukunaga et al., Cell 61:341-350 [1990a]; Fukunaga et al. Proc. Natl.Acad. Sci. USA 87:8702-8706 [1990b]; Larsen et al. J. Exp. Med.172:1559-1570 [1990]), erythropoietin (EPO) (D'Andrea et al., Cell57:277-285 [1989]; Jones et al., Blood 76:31-35 [1990]), Leukemiainhibitory factor (LIF) (Gearing et al., EMBO J. 10:2839-2848 [1991]),oncostatin M (OSM) (Rose et al., Proc. Natl. Acad. Sci. USA 88:8641-8645[1991]) and also receptors for prolactin (Boutin et al., Proc. Natl.Acad. Sci. USA 88:7744-7748 [1988]; Edery et al., Proc. Natl. Acad. Sci.USA 86:2112-2116 [1989]), growth hormone (GH) (Leung et al., Nature330:537-543 [1987]) and ciliary neurotrophicfactor (CNTF) (Davis et al.,Science 253:59-63 [1991].

Members of the cytokine receptor superfamily may be grouped into threefunctional categories (for review see Nicola et al., Cell 67:1-4[1991]). The first class comprises single chain receptors, such aserythropoietin receptor (EPO-R) or granulocyte colony stimulating factorreceptor (G-CSF-R) bind ligand with high affinity via the extracellulardomain and also generate an intracellular signal. A second class ofreceptors, so call α-subunits, includes interleukin-6 receptor (IL6-R),granulocyte-macrophage colony stimulating factor receptor (GM-CSF-R),interleukin-3 receptor (IL3-Rα) and other members of the cytpkinereceptor superfamily. These α-subunits bind ligand with low affinity butcannot transduce an intracellular signal. A high affinity receptorcapable of signaling is generated by a heterodimer between an α-subunitand a member of a third class of cytokine receptors, termed β-subunits,e.g. β_(c), the common β-subunit for the three α-subunits IL3-Rα andGM-CSF-R.

Evidence that mpl is a member of the cytokine receptor superfamily comesfrom sequence homology (Gearing, D. P., EMBO J. 8:3667-3676 [1988];Bazan, J. F., Proc. Natl. Acad. Sci. USA 87:6834-6938 [1990]; Davis S.,et al., Science 253:59-63 [1991] and Vigon et al., Proc. Natl. Acad.Sci. USA 89:5640-5644 [1992]) and its ability to transduce proliferativesignals.

Deduced protein sequence from molecular cloning of murine c-mpl revealsthis protein is homologous to other cytokine receptors. Theextracellular domain contains 465 amino acid residues and is composed oftwo subdomains each with four highly conserved cysteines and a WGXWSmotif in the N-terminal subdomain and WSXWS motif in the C-terminalsubdomain. The ligand-binding extracellular domains are predicted tohave similar double β-barrel fold structural geometries. This duplicatedextracellular domain is highly homologous to the signal transducingchain common to IL-3, IL-5 and GM-CSF receptors as well as thelow-affinity binding domain of LIF (Vigon I., et al., Oncogene8:2607-2615 [1993]). Thus mpl may belong to the low affinity ligandbinding class of cytokine receptors.

The extracellular domain is followed by a 22 residue transmembranedomain and a 121 residue cytoplasmic domain rich in serine and proline.The cytoplasmic domain contains no consensus protein kinase, phosphotaseor any other known motif associated with signal transduction.

A comparison of murine mpl and mature human mpl P, reveals these twoproteins show 81% sequence identity. More specifically, the N-terminusand C-terminus extracellular subdomains share 75% and 80% identityrespectively. The most conserved mpl region is the cytoplasmic domainshowing 91% amino acid identity, with a sequence of 37 residues near thetransmembrane domain being identical in both species. Accordingly, mplis reported to be one of the most conserved members of the cytokinereceptor superfamily (Vigon supra).

Evidence that mpl is a functional receptor capable of transducing aproliferative signal comes from construction of chimeric receptorscontaining an extracellular domain from a cytokine receptor having highaffinity for a known cytokine with the mpl cytoplasmic domain. Since noknown ligand for mpl has been reported, it was necessary to constructthe chimeric high affinity ligand binding extracellular domain from aclass one cytokine receptor such as IL-4R or G-CSFR. Vigon et al. suprafused the extracellular domain of G-CSFR with both the transmembrane andcytoplasmic domain of c-mpl. An IL-3 dependent cell line, BAF/B03 wastransfected with the G-CSFR/mpl chimera along with a full length G-CSFRcontrol. Cells transfected with the chimera grew equally well in thepresence of cytokine IL-3 or G-CSF. Similarly, cells transfected withG-CSFR also grew well in either IL-3 or G-CSF. All cells died in theabsence of growth factors. A similar experiment was conducted by Skoda,R. C. et al. EMBO J. 12(7):2645-2653 (1993) in which both theextracellular and transmrembrane domains of human IL4 receptor (hIL4-R)were fused to the murine mpl cytoplasmic domain, and transfected into amurine IL3 dependent Ba/F3 cell line. Ba/F3 cells transfected withwildtype hIL4-R proliferated normally in either the presence of thespecies specific IL-4 or IL-3. BaF3 cells transfected with hIL4R/mplproliferated normally in the presence of hIL4 (in the presence orabsence of IL3) demonstrating that in Ba/F3 cells the mpl cytoplasmicdomain contains all the elements necessary to transduce a proliferativesignal.

These chimeric experiments demonstrate the proliferation signalingcapability of the mpl extracellular domain but are silent regardingwhether the mpl extracellular domain can bind a ligand. These resultsare consistent with at least two possibilities, namely, mpl is a singlechain (class one) receptor like EpoR or G-CSFR or it is a signaltransducing β-subunit (class three) requiring an α-subunit like IL-3(Skoda et al. supra).

V. Mpl Ligand Stimulates Megakaryocytopoiesis

As described above, it has been suggested that serum contains a uniquefactor, sometimes referred to as thrombopoietin, that actssynergistically with various other cytokines to promote growth andmaturation of megakaryocytes. No such natural factor has ever beenisolated from serum or any other source even though considerable efforthas been expended by numerous groups. Even though it is not know whethermpl is capable of directly binding a megakaryocyte stimulating factor,recent experiments demonstrate that mpl is involved in proliferativesignal transduction from a factor or factors found in the serum ofpatients with aplastic bone marrow (Methia, N. et al., Blood82(5):1395-1401 [1993]).

Evidence that a unique serum colony-forming factor distinct from IL-1α,IL-3, IL-4, IL-6, IL-11, SCF, EPO, G-CSF, and GM-CSF transduces aproliferative signal through mpl comes from examination of thedistribution of c-mpl expression in primitive and committedhemotopoietic cell lines and from mpl antisense studies in one of thesecell lines.

Using reverse transcriptase (RT)-PCR in immuno-purified, humanhematopoietic cells Methia et al. supra demonstrated that strong mplmRNA messages were only found in CD34+ purified cells, megakaryocytesand platelets. CD34+ cells purified from Bone Marrow (BM) representsabout 1% of all BM cells and are enriched in primitive and committedprogenitors of all lineages (e.g. erythroid, granulomacrophage, andmegakaryocytic).

Mpl antisense oligodeoxy nucleotides were shown to suppressmegakaryocytic colony formation from the pluripotent CD34+ cellscultured in serum from patients with aplastic marrow (a rich source ofmegakaryocytes stimulating activity [Meg-CSA]). These same antisenseoligodeoxynucleotides had no effect on erythroid or granulomacrophagecolony formation.

Whether mpl directly binds a ligand and whether the serum factor shownto cause megakaryocytopoiesis acts through mpl is still unknown. It hasbeen suggested, however, that if mpl does directly bind a ligand, itsamino acid sequence is likely to be highly conserved and have speciescross reactivity owing to the considerable sequence identity betweenhuman and murine mpl extracellular domain (Vigon et al., supra[1993]).

In view of the foregoing it will be appreciated there is a current andcontinuing need in the art to isolate and identify molecules capable ofstimulating differentiation and maturation of megakaryocytes fortherapeutic use in the treatment of thrombocytopenia. It is believedsuch a molecule is a mpl ligand and thus there exists a further need toisolate such ligand(s) to evaluate their role(s) in cell growth anddifferentiation.

Accordingly, it is an object of this invention to obtain a novelpharmaceutically pure molecule capable of stimulating differentiationand maturation of megakaryocytes into the mature platelet-producingform.

It is another object to provide the novel molecule in a form fortherapeutic use in the treatment of thrombocytopenia.

It is a further object of the present invention to isolate, purify andspecifically identify novel protein ligands capable of binding in vivo acytokine superfamily receptor known as mpl and to transduce aproliferative signal.

It is still another object to provide novel nucleic acid moleculesencoding such protein ligands and to use this nucleic acid molecule toproduce mpl binding ligands in recombinant cell culture for diagnosticand therapeutic use.

It is yet another object to provide derivatives and modified forms ofthe protein ligands including amino acid sequence variants, novelglycoprotein forms and other covalent derivatives thereof.

It is an additional object to provide fusion polypeptide forms combininga novel mpl ligand and a heterologous protein and covalent derivativesthereof.

It is yet an additional object to prepare immunogens for raisingantibodies against novel mpl ligands or fusion forms thereof, as well asto obtain antibodies capable of binding such ligands.

These and other objects of the invention will be apparent to theordinary artisan upon consideration of the specification as a whole.

SUMMARY OF THE INVENTION

The objects of the invention are achieved by providing an isolatedmammalian megakaryocytopoietic maturation promoting protein capable ofstimulating maturation and/or differentiation of megakaryocytes into themature platelet-producing form. This substantially homogeneous proteindenominated the “mpl ligand” is purified from a natural source by theprocedures described herein and has the following characteristics:

(1) The partially purified ligand isolated from aplastic porcine plasmaelutes from a gel filtration run in either PBS, PBS containing 0.1% SDSor PBS containing 4M MgCl₂ with Mr of 60,000-70,000;

(2) The ligand's activity is destroyed by pronase;

(3) The ligand is stable to low pH (2.5), SDS to 0.1%, and 2M urea;

(4) The ligand is a glycoprotein, based on its binding to a variety oflectin columns;

(5) The highly purified ligand elutes from non-reduced SDS-PAGE with aMr of 25,000-35,000. Smaller amounts of activity also elute with Mr of˜18,000 and 60,000;

(6) The highly purified ligand resolves on reduced SDS-PAGE as a doubletwith Mr of 28,000 and 31,000;

(7) The amino-terminal sequence of the 18,000, 28,000 and 31,000 bandsis the same—SPAPPACDPRLLNKLLRDDH SEQ ID NO.: 1); and

(8) The ligand binds and elutes from the following affinity columns

Blue-Sepharose,

CM Blue-Sepharose,

MONO-Q,

MONO-S,

Lentil lectin-Sepharose,

WGA-Sepharose,

CON A-Sepharose,

Ether 650 m Toyopearl,

Butyl 650 m Toyopearl,

Phenyl 650 m Toyopearl, and

Phenyl-Sepharose.

The “mpl ligand” polypeptide of this invention preferably has at least80% sequence identity with the amino acid sequence of the highlypurified mpl ligand isolated from aplastic porcine plasma describedherein. Most preferably the mpl ligand of this invention is mature humanmpl ligand or a protein having 80% sequence identity with mature humanmpl ligand. Optionally the mpl ligand polypeptide or fragment thereofmay be fused to a heterologous polypeptide. A preferred heterologouspolypeptide is a cytokine.

Another aspect of this invention provides a composition comprising anisolated mpl ligand capable of stimulating the incorporation of labelednucleotides (³H-thymidine) into the DNA of IL-3 dependent Ba/F3 cellstransfected with human mpl P and isolated from its source environmentsufficiently free of contaminating source proteins to yield anunambiguous N-terminus amino acid sequence of at least 20 residues. Inone embodiment, the N-terminus amino acid sequence of this ligand isSPAPPACDPRLLNKLLRDDH (SEQ ID NO.: 1). Optionally, the ligand of thisinvention has a portion of its amino acid sequence at or near itsN-terminus that has at least 80% sequence identity with the sequence;SPAPPACDPRLLNKLLRDDH (SEQ ID NO.:1).

In still another aspect, a method is provided for purifying mpl ligandmolecules comprising contacting a source plasma containing the mplligand molecules to be purified with an immobilized receptorpolypeptide, specifically mpl or a mpl fusion polypeptide immobilized ona support, under conditions whereby the mpl ligand molecules to bepurified are selectively adsorbed onto the immobilized receptorpolypeptide, washing the immobilized support to remove non-adsorbedmaterial, and eluting the molecules to be purified from the immobilizedreceptor polypeptide to which they are adsorbed with an elution buffer.Preferably the source plasma containing the mpl ligand is aplasticporcine plasma and the immobilized receptor is a mpl-IgG fusion. Alsopreferably the immobilized support is washed with PBS/PBS in 2M NaCl andthe elution buffer is 0.1M glycine-HCl, pH 2.25.

In another embodiment, this invention provides an isolated antibodycapable of binding to the mpl ligand. The isolated antibody capable ofbinding to the mpl ligand may optionally be fused to a secondpolypeptide and the antibody or fusion thereof may be used to isolateand purify mpl ligand from a source as described above for immobilizedmpl polypeptide. In a further aspect of this embodiment, the inventionprovides a method for detecting the mpl ligand in vitro or in vivocomprising contacting the antibody with a sample, especially a serumsample, suspected of containing the ligand and detecting if binding hasoccurred.

In still further embodiments, the invention provides an isolated nucleicacid molecule, encoding the mpl ligand or fragments thereof, whichnucleic acid molecule may be labeled or with a detectable moiety, and anucleic acid molecule having a sequence that is complementary to, orhybridizes under stringent or moderately stringent conditions with, anucleic acid molecule having a sequence encoding a mpl ligand. In afurther aspect of this embodiment, the nucleic acid molecule is DNAencoding the mpl ligand and further comprises a replicable vector inwhich the DNA is operably linked to control sequences recognized by ahost transformed with the vector. This aspect further includes hostcells transformed with the vector and a method of using the DNA toeffect production of mpl ligand, comprising expressing the DNA encodingthe mpl ligand in a culture of the transformed host cells and recoveringthe mpl ligand from the host cells or the host cell culture. The mplligand prepared in this manner is preferably human mpl ligand.

The invention further includes a method for treating a mammal havingthrombocytopenia comprising administering a therapeutically effectiveamount of a mpl Ligand to the mammal. Optionally the mpl Ligand isadministered in combination with a cytokine, especially a colonystimulating factor or interleukin. Preferred colony stimulating factorsor interleukins include; G-CSF, CSF-1, GM-CSF, M-CSF, erythropoietin,IL-1, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, or IL-11.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Effect of pronase, DTT and heat on the ability of APP tostimulate Ba/F3-mpl cell proliferation. For prohase digestion of APP,pronase (Boehringer Mannheim) or bovine serum albumin was coupled toaffi-gel10 (Biorad) and incubated individually with APP for 18 hrs. at37 C. Subsequently, the resins were removed by centrifugation andsupernatants asayed. APP was also heated to 80 C. for 4 min. or made 100uM DTT followed by dialysis against PBS.

FIG. 2. Elution of mpl-ligand activity from Phenyl-Toyopearl,Blue-Sepharose and Ultralink-mpl columns. Fractions 4-8 from the mplaffinty column were the peak activity fractions eluted from the column.

FIG. 3. SDS-PAGE of eluted Ultralink-mpl fractions. To 200 ul of eachfraction 2-8, 1 ml of acetone containing 1 mM HCl at −20 C. was added.After 3 hrs. at −20 C. samples were centrifuged and resultant pelletswere washed 2× with acetone at −20 C. The acetone pellets weresubsequently dissolved in 30 ul of SDS-solubilization buffer, made 100uM DTT and heated at 90 C. for 5 min. The samples were then resolved ona 4-20% SDS-polyacrlyamide gel and proteins were visualized by silverstaining.

FIG. 4. Elution of mpl-ligand activity from SDS-PAGE. Fraction 6 fromthe mpl-affinity column was resolved on a 4-20% SDS-polyacrylamide gelunder non-reducing conditions. Following electrophoresis the gel wassliced into 12 equal regions and electroeluted as described in the text.The electroeluted samples were dialyzed into PBS and assayed at a 1/20dilution. The Mr standards used to calibrate the gel were Novex Mark 12standards.

FIG. 5. Effect of mpl-ligand depleted APP on human megakaryocytopoiesis.mpl-Ligand depleted APP was made by passing 1 ml over a 1 mlmpl-affinity column (700 ug mpl-IgG/ml NHS-superose, Pharmacia). Humanperipheral stem cell cultures were made 10% APP or 10% mpl-liganddepleted APP and cultured for 12 days. Megakaryocytopoiesis wasquantitated as described in the text.

FIG. 6. Effect of mpl-IgG on the stimulation of humanmegakaryocytopoiesis by APP. Human peripheral stem cell cultures weremade 10% with APP and cultured for 12 days. At day 0, 2 and 4, mpl-IgG(0.5 ug) or ANP-R-IgG (0.5 ug) was added. After 12 daysmegakaryocytopoiesis was quantitated as described in the text. Theaverage of duplicate samples is graphed with the actual duplicate datain parenthesis.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

In general, the following words or phrases have the indicated definitionwhen used in the description, examples, and claims.

“Cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormone,insulin-like growth factors, human growth hormone, N-methionyl humangrowth hormone, bovine growth hormone, parathyroid hormone, thyroxine,insulin, proinsulin, relaxin, prorelaxin, glycoprotein hormones such asfollicle stimulating hormone (FSH), thyroid stimulating hormone (TSH),and leutinizing hormone (LH), hemopoietic growth factor, hepatic growthfactor, fibroblast growth factor, prolactin, placental lactogen, tumornecrosis factor-alpha and -beta, mullerian-inhibiting substance, mousegonadotropin-associated peptide, inhibin, activin, vascular endothelialgrowth factor, integrin, thrombopoietin, nerve growth factors such asNGF-β, platelet-growth factor, transforming growth factors (TGFs) suchas TGF-alpha and TGF-beta, insulin-like growth factor-I and -II,erythropoietin, osteoinductive factors, interferons such asinterferon-alpha, -beta, and -gamma, colony stimulating factors (CSFs)such as macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), andgranulocyte-CSF (G-CSF), interleukins (ILs) such as IL-1, IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-8 and other polypeptide factors. As usedherein the foregoing terms are meant to include proteins from naturalsources or from recombinant cell culture. Similarly, the terms areintended to include biologically active equivalents; e.g. differing inamino acid sequence by one or more amino acids or in type or extent ofglycosylation.

A “mpl ligand” is any polypeptide that possesses the property of bindingto mpl, a member of the cytokine receptor superfamily, and having abiological property of the mpl ligand defined below. The preferredbiological property is the ability to stimulate the incorporation oflabeled nucleotides (e.g. ³H-thymidine) into the DNA of IL-3 dependentBa/F3 cells transfected with human mpl P. This definition encompassesthe poylpeptide isolated from aplastic porcine plasma described hereinor from another source and includes functional derivatives, fragments,alleles, isoforms and analogus thereof.

“Isolated mpl ligand”, “highly purified mpl ligand” and “substantiallyhomogenous mpl ligand” are used interchangeably and mean a mpl ligandthat has been purified from a mpl ligand source and is sufficiently freeof other source proteins (1) to obtain at least 15 and preferably 20amino acid residues of the N-terminal or of an internal amino acidsequence by using a spinning cup sequenator or the best commerciallyavailable amino acid sequenator marketed or as modified by publishedmethods as of the filing date of this application, or (2) to homogeneityby SDS-PAGE under non-reducing or reducing conditions using Coomassieblue or, preferably, silver stain. Homogeneity here means less then 5%contaminated with other source proteins.

“Biological property” when used in conjunction with either the “mplligand” or “Isolated mpl ligand” means an in vivo effector or antigenicfunction or activity that is directly or indirectly caused or performedby a mpl ligand (whether in its native or denatured conformation) or afragment thereof. Effector functions include mpl binding and any carrierbinding activity, agonism or antagonism of mpl, especially transductionof a proliferative signal including replication, DNA regulatoryfunction, modulation of the biological activity of other cytokines,receptor (especially cytokine) activation, deactivation, up- or downregulation, cell growth or differentiation and the like. An antigenicfunction means possession of an epitope or antigenic site that iscapable of cross-reacting with antibodies raised against the mpl ligand.

“Biologically active” when used in conjunction with either the “mplligand” or “Isolated mpl ligand” means a mpl ligand or polypeptide thatshares an effector and/or antigenic function of the mpl ligand isolatedfrom aplastic porcine plasma described herein. A principal knowneffector function of the mpl ligand or polypeptide herein is binding tompl and stimulating the incorporation of labeled nucleotides(³H-thymidine) into the DNA of IL-3 dependent Ba/F3 cells transfectedwith human mpl P. The principal antigenic function of a mpl ligandpolypeptide is that it binds with an affinity of at least about 10⁶l/mole to an antibody raised against the mpl ligand isolated fromaplastic porcine plasma. Ordinarily, the polypeptide binds with anaffinity of at least about 10⁷ l/mole. Most preferably, theantigenically active mpl ligand polypeptide is a polypeptide that bindsto an antibody raised against the mpl ligand having one of the abovedescribed effector functions. The antibodies used to define“biologically activity” are rabbit poly clonal antibodies raised byformulating the mpl ligand isolated from aplastic porcine plasma inFreund's complete adjuvant, subcutaneously injecting the formulation,and boosting the immune response by intraperitoneal injection of theformulation until the titer of mpl ligand antibody plateaus.

“Percent amino acid sequence Identity” with respect to the mpl ligandsequence is defined herein as the percentage of amino acid residues inthe candidate sequence that are identical with the residues in the mplligand sequence isolated from aplastic porcine plasma after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. None of N-terminal,C-terminal, or internal extensions, deletions, or insertions into thempl ligand sequence isolated from aplastic porcine plasma shall beconstrued as affecting sequence identity or homology. Thus exemplarybiologically active mpl ligand polypeptides considered to have identicalsequences include; prepro-mpl ligand, pro-mpl ligand, and mature mplligand.

“Mpl ligand microsequencing” may be accomplished by any appropriatestandard procedure provided the procedure is sensitive enough. In onesuch method, highly purified polypeptide obtained from SDS gels or froma final HPLC step are sequenced directly by automated Edman (phenylisothiocyanate) degradation using a model 470A Applied Biosystems gasphase sequencer equipped with a 120A phenylthiohydantion (PTH) aminoacid analyzer. Additionally, mpl ligand fragments prepared by chemical(e.g. CNBr, hydroxylamine, 2-nitro-5-thiocyanobenzoate) or enzymatic(e.g. trypsin, clostripain, staphylococcal protease) digestion followedby fragment purification (e.g. HPLC) may be similarly sequenced. PTHamino acids are analyzed using the ChromPerfect data system (JusticeInnovations, Palo Alto, Calif.). Sequence interpretation is performed ona VAX 11/785 Digital Equipment Co. computer as described by Henzel etal.,J. Chromatography 404:41-52 (1987). Optionally, aliquots of HPLCfractions may be electrophoresed on 5-20% SDS-PAG, electrotransfered toa PVDF membrane ProBlott, AIB, Foster City, Calif.) and stained withCoomassie Brilliant Blue (Matsudiara, P., J. Biol. Chem. 262:10035-10038(1987). A specific protein identified by the stain is excised from theBlot and N-terminal sequencing is carried out with the gas phasesequenator described above. For internal protein sequences, HPLCfractions are dried under vacuum (SpeedVac), resuspended in appropriatebuffers, and digested with cyanogen bromide, the Lys-specific enzymeLys-C (Wako Chemicals, Richmond, Va.), or Asp-N (Boehringer Mannheim,Indianapolis, Ind.). After digestion, the resultant peptides aresequenced as a mixture or after HPLC resolution on a C4 column developedwith a propanol gradient in 0.1% TFA prior to gas phase sequencing.

“Mpl ligand variants” or “Mpl ligand sequence variants” as definedherein means a biologically active mpl ligand as defined above havingless than 100% sequence identity with the mpl ligand isolated fromaplastic porcine plasma described herein. Ordinarily, a biologicallyactive mpl ligand variant will have an amino acid sequence having atleast 70% amino acid sequence identity with the mpl ligand isolated fromaplastic porcine plasma, preferably at least 75%, more preferably atleast 80%, still more preferably at least 85%, even more preferably atleast 90%, and most preferably at least 95%.

“Mpl ligand fragments” as used herein have a consecutive sequence of atleast 10, 15, 20, 25, 30, or 40 amino acid residues that are identicalto the sequences of the mpl ligand isolated from aplastic porcine plasmadescribed herein. An example of a mpl ligand fragment is the N-terminaldomain (“NTD”) having the sequence SPAPPACDPRLLNKLLRDDHVLHGR SEQ ID NO.:2). Other examples of mpl ligand fragments include those produced as aresult of chemical or enzymatic digestion of the purified ligand asdescribed above under “Mpl ligand microsequencing”.

“Thrombocytopenia” is defined as a platelet count below 150×10⁹ perliter of blood.

“Thrombopoeitic activity” is defined as biological activity thatconsists of accelerating the differentiation and/or maturation ofmegakaryocytes or megakaryocyte precursors into the platelet producingform of these cells. This activity may be measured in various assaysincluding an in-vivo mouse platelet rebound synthesis assay, inductionof platelet cell surface antigen assay as measured by an anti-plateletimmunoassay (anti-GPII_(b)III_(a)) for a human leukemia megakaryoblasticcell line (CMK), and induction of polyploidization in a megakaryoblasticcell line(DAMI).

“Thrombopoeitin” (TPO) is defined as a compound having Thrombopoeiticactivity or being capable of increasing serum platelet counts in amammal. TPO is preferably capable of increasing endogenous plateletcounts by at least 10%, more preferably by 50%, and most preferablycapable of elevating platelet counts in a human to greater that 150×10⁹per liter of blood.

“Isolated mpl ligand nucleic ac id” is RNA or DNA containing greaterthan 16, preferably 20 or more, sequential nucleotide bases that encodebiologically active mpl ligand or a fragment thereof, is complementaryto the RNA or DNA, or hybridizes to the RNA or DNA and remains stablybound under stringent conditions. This RNA or DNA is free from at leastone contaminating source nucleic acid with which it is normallyassociated in the natural source and preferably substantially free ofany other mammalian RNA or DNA. The phrase “free from at least onecontaminating source nucleic acid with which it is normally associated”includes the case where the nucleic acid is present in the source ornatural cell but is in a different chromosomal location or is otherwiseflanked by neucleic acid sequences not normally found in the sourcecell. An example of isolated mpl ligand nucleic acid is RNA or DNA thatencodes a biologically active mpl ligand sharing at least 75% sequenceidentity, more preferably at least 80%, still more preferably at least85%, even more preferably 90%, and most preferably 95% sequence identitywith the porcine mpl ligand.

“Control sequences” when referring to expression means DNA sequencesnecessary for the expression of an operably linked coding sequence in aparticular host organism. The control sequences that are suitable forprokaryotes, for example, include a promoter, optionally an operatorsequence, a ribosome binding site, and possibly, other as yet poorlyunderstood sequences. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

“Operably linked” when referring to nucleic acids means that the nucleicacids are placed in a functional relationship with another nucleic acidsequence. For example, DNA for a presequence or secretory leader isoperably linked to DNA for a polypeptide if it is expressed as apreprotein that participates in the secretion of the polypeptide; apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence; or a ribbsome binding site isoperably linked to a coding sequence if it is positioned so as tofacilitate translation. Generally, “operably linked” means that the DNAsequences being linked are contiguous and, in the case of a secretoryleader, contiguous and in reading phase. However, enhancers do not haveto be contiguous. Linking is accomplished by ligation at convenientrestriction sites. If such sites do not exist, the syntheticoligonucleotide adaptors or linkers are used in accord with conventionalpractice.

“Exogenous” when referring to an element means a nucleic acid sequencethat is foreign to the cell, or homologous to the cell but in a positionwithin the host cell nucleic acid in which the element is ordinarily notfound.

“Cell,” “cell line,” and “cell culture” are used interchangeably hereinand such designations include all progeny of a cell or cell line. Thus,for example terms like “transformants” and “transformed cells” includethe primary subject cell and cultures derived therefrom without regardfor the number of transfers. It is also understood that all progeny maynot be precisely identical in DNA content, due to deliberate orinadvertent mutations. Mutant progeny that have the same function orbiological activity as screened for in the originally transformed cellare included. Where distinct designations are intended, it will be clearfrom the context.

“Plasmids” are autonomously replicating circular DNA moleculespossessing independent origins of replication and are designated hereinby a lower case “p” preceded and/or followed by capital letters and/ornumbers. The starting plasmids herein are either commercially available,publicly available on an unrestricted basis, or can be constructed fromsuch available plasmids in accord with published procedures. Inaddition, other equivalent plasmids are known in the art and will beapparent to the ordinary artisan.

“Restriction enzyme digestion” when referring to DNA means catalyticcleavage of internal phosphodiester bonds of DNA with an enzyme thatacts only at certain locations or sites in the DNA sequence. Suchenzymes are called “restriction endonucleases”. Each restrictionendonuclease recognizes a specific DNA sequence called a “restrictionsite” that exhibits twofold symmetry. The various restriction enzymesused herein are commercially available and their reaction conditions,cofactors, and other requirements as established by the enzyme suppliersare used. Restriction enzymes commonly are designated by abbreviationscomposed of a capital letter followed by other letters representing themicroorganism from which each restriction enzyme originally was obtainedand then a number designating the particular enzyme. In general, about 1μg of plasmid or DNA fragment is used with about 1-2 units of enzyme inabout 20 μl of buffer solution. Appropriate buffers and substrateamounts for particular restriction enzymes are specified by themanufacturer. Incubation of about 1 hour at 37° C. is ordinarily used,but may vary in accordance with the supplier's instructions. Afterincubation, protein or polypeptide is removed by extraction with phenoland chloroform, and the digested nucleic acid is recovered from theaqueous fraction by precipitation with ethanol. Digestion with arestriction enzyme may be followed with bacterial alkaline phosphatasehydrolysis of the terminal 5′ phosphates to prevent the tworestriction-cleaved ends of a DNA fragment from “circularizing” orforming a closed loop that would impede insertion of another DNAfragment at the restriction site. Unless otherwise stated, digestion ofplasmids is not followed by 5′ terminal dephosphorylation. Proceduresand reagents for dephosphorylation are conventional as described insections 1.56-1.61 of Sambrook et al., Molecular Cloning: A LaboratoryManual [New York: Cold Spring Harbor Laboratory Press, 1989].

“Recovery” or “isolation” of a given fragment of DNA from a restrictiondigest means separation of the digest on polyacrylamide or agarose gelby electrophoresis, identification of the fragment of interest bycomparison of its mobility versus that of marker DNA fragments of knownmolecular weight, removal of the gel section containing the desiredfragment, and separation of the gel from DNA. This procedure is knowngenerally. For example, see Lawn et al., Nucleic Acids Res., 9:6103-6114(1981), and Goeddel et al., Nucleic Acids Res. 8:4057 (1980).

“Southern analysis” or “Southern blotting” is a method by which thepresence of DNA sequences in a restriction endonuclease digest of DNA orDNA-containing composition is confirmed by hybridization to a known,labeled oligonucleotide or DNA fragment. Southern analysis typicallyinvolves electrophoretic separation of DNA digests on agarose gels,denaturation of the DNA after electrophoretic separation, and transferof the DNA to nitrocellulose, nylon, or another suitable membranesupport for analysis with a radiolabeled, biotinylated, orenzyme-labeled probe as described in sections 9.37-9.52 of Sambrook etal., supra.

“Northern analysis” or “Northern blotting” is a method used to identifyRNA sequences that hybridize to a known probe such as anoligonucleotide, DNA fragment, cDNA or fragment thereof, or RNAfragment. The probe is labeled with a radioisotope such as 32-P, or bybiotinylation, or with an enzyme. The RNA to be analyzed is usuallyelectrophoretically separated on an agarose or polyacrylamide gel,transferred to nitrocellulose, nylon, or other suitable membrane, andhybridized with the probe, using standard techniques well known in theart such as those described in sections 7.39-7.52 of Sambrook et al.,supra.

“Ligation” is the process of forming phosphodiester bonds between twonucleic acid fragments. For ligation of the two fragments, the ends ofthe fragments must be compatible with each other. In some cases, theends will be directly compatible after endonuclease digestion. However,it may be necessary first to convert the staggered ends commonlyproduced after endonuclease digestion to blunt ends to make themcompatible for ligation. For blunting the ends, the DNA is treated in asuitable buffer for at least 15 minutes at 15° C. with about 10 units ofthe Klenow fragment of DNA polymerase I or T4 DNA polymerase in thepresence of the four deoxyribonucleotide triphosphates. The DNA is thenpurified by phenol-chloroform extraction and ethanol precipitation. TheDNA fragments that are to be ligated together are put in solution inabout equimolar amounts. The solution will also contain ATP, ligasebuffer, and a ligase such as T4 DNA ligase at about 10 units per 0.5 μgof DNA. If the DNA is to be ligated into a vector, the vector is firstlinearized by digestion with the appropriate restrictionendonuclease(s). The linearized fragment is then treated with bacterialalkaline phosphatase or calf intestinal phosphatase to preventself-ligation during the ligation step.

“Preparation” of DNA from cells means isolating the plasmid DNA from aculture of the host cells. Commonly used methods for DNA preparation arethe large- and small-scale plasmid preparations described in sections1.25-1.33 of Sambrook et al., supra. After preparation of the DNA, itcan be purified by methods well known in the art such as that describedin section 1.40 of Sambrook et al., supra.

“Oligonucleotides” are short-length, single- or double-strandedpolydeoxynucleotides that are chemically synthesized by known methods(such as phosphotriester, phosphite, or phosphoramidite chemistry, usingsolid-phase techniques such as described in EP 266,032 published May 4,1988, or via deoxynucleoside H-phosphonate intermediates as described byFroehler et al., Nucl. Acids Res., 14: 5399-5407 [1986]. Further methodsinclude the polymerase chain reaction defined below and other autoprimermethods and oligonucleotide syntheses on solid supports. All of thesemethods are described in Engels et al., Agnew. Chem. Int. Ed. Engl., 28:716-734 [1989]). These methods are used if the entire nucleic acidsequence of the gene is known, or the sequence of the nucleic acidcomplementary to the coding strand is available. Alternatively, if thetarget amino acid sequence is known, one may infer potential nucleicacid sequences using known and preferred coding residues for each aminoacid residue. The oligonucleotides are then purified on polyacrylamidegels.

“Polymerase chain reaction” or “NPCR” refers to a procedure or techniquein which minute amounts of a specific piece of nucleic acid, RNA and/orDNA, are amplified as described in U.S. Pat. No. 4,683,195 issued Jul.28, 1987. Generally, sequence information from the ends of the region ofinterest or beyond needs to be available, such that oligonucleotideprimers can be designed; these primers will be identical or similar insequence to opposite strands of the template to be amplified. The 5′terminal nucleotides of the two primers may coincide with the ends ofthe amplified material. PCR can be used to amplify specific RNAsequences, specific DNA sequences from total genomic DNA, and cDNAtranscribed from total cellular RNA, bacteriophage or plasmid sequences,etc. See generally Mullis et al., Cold Spring Harbor Symp. Quant. Biol.,51: 263 (1987); Erlich, ed., PCR Technology, (Stockton Press, NY, 1989).As used herein, PCR is considered to be one, but not the only, exampleof a nucleic acid polymerase reaction method for amplifying a nucleicacid test sample comprising the use of a known nucleic acid as a primerand a nucleic acid polymerase to amplify or generate a specific piece ofnucleic acid.

“Stringent conditions” are those that (1) employ low ionic strength andhigh temperature for washing, for example, 0.015 M NaCl/0.0015 M sodiumcitrate/0.1%. NaDodSO₄ at 50° C., or (2) employ during hybridization adenaturing agent such as formamide, for example, 50% (vol/vol) formamidewith 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodiumcitrate at 42° C. Another example is use of 50% formamide, 5×SSC (0.75 MNaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1%sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA(50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at42° C. in 0.2×SSC and 0.1% SDS.

II. Preferred Embodiments of the Invention

Preferred polypeptides of this invention are substantially homogeneouspolypeptide(s), refered to as mpl ligand(s), that possesses the propertyof binding to mpl, a member of the receptor cytokine superfamily, andhaving the biological property of stimulating the incorporation oflabled nucleotides (³H-thymidine) into the DNA of IL-3 dependant Ba/F3cells transfected with human mpl P. More preferred mpl ligand(s) areisolated mammalian protein(s) having thrombopoeitic activity, namely,being capable of stimulating maturation and/or differentiation ofimmature megakaryocytes into the mature platelet-producing form. Mostpreferred polypeptides of this invention are human mpl ligand(s) havingthrombopoeitic activity. Optionally these human mpl ligand(s) lackglycosylation.

Optional preferred polypeptides of this invention are biologicallyactive mpl ligand variant(s) that have an amino acid sequence having atleast 70% amino acid sequence identity with the mpl ligand isolated fromaplastic porcine plasma, preferably at least 75%, more preferably atleast 80%, still more preferably at least 85%, even more preferably atleast 90%, and most preferably at least 95%. The mpl ligand isolatedfrom aplastic porcine plasma has the following characteristics:

(1) The partially purified ligand isolated from aplastic porcine plasmaelutes from a gel filtration run in either PBS, PBS containing 0.1% SDSor PBS containing 4M MgCl₂ with Mr of 60,000-70,000;

(2) The ligand's activity is destroyed by pronase;

(3) The ligand is stable to low pH (2.5), SDS to 0.1%, and 2M urea;

(4) The ligand is a glycoprotein, based on its binding to a variety oflectin columns;

(5) The highly purified-ligand elutes from non-reduced SDS-PAGE with aMr of 25,000-35,000. Smaller amounts of activity also elute with Mr of˜18,000 and 60,000;

(6) The highly purified ligand resolves on reduced SDS-PAGE as a doubletwith Mr of 28,000 and 31,000;

(7) The amino-terminal sequence of the 18,000, 28,000 and31,000 bands isthe same—SPAPPACDPRLLNKLLRDDHVLHGR (SEQ ID NO.: 2); and

(8) The ligand binds and elutes from the following affinity columns

Blue-Sepharose,

CM Blue-Sepharose,

MONO-Q,

MONO-S,

Lentil lectin-Sepharose,

WGA-Sepharose,

CON A-Sepharose,

Ether 650 m Toyopearl,

Butyl 650 m Toyopearl,

Phenyl 650 m Toyopearl, and

Phenyl-Sepharose.

Other preferred naturally occuring biologically active mpl ligandpolypeptides of this invention include; prepro-mpl ligand, pro-mplligand, mature mpl ligand and glycosylation variants thereof.

Still other preferred polypeptides of this invention include Mpl ligandsequence variants. Ordinarily, preferred Mpl ligand sequence variantsare biologically active mpl ligand variants that have an amino acidsequence having at least 70% amino acid sequence identity with the mplligand isolated from aplastic porcine plasma, preferably at least 75%,more preferably at least 80%, still more preferably at least 85%, evenmore preferably at least 90%, and most preferably at least 95%. Anexemplary preferred variant is a fusion between mpl ligand or fragment(defined below) thereof and another cytokine or fragment thereof.

Other preferred polypeptides of this invention include Mpl ligandfragments having a consecutive sequence of at least 10, 15, 20, 25, 30,or 40 amino acid residues that are identical to the sequences of the mplligand isolated from aplastic porcine plasma described herein. Apreferred mpl ligand fragment is the N-terminal domain (“NTD”) havingthe sequence SPAPPACDPRLLNKLLRDDHVLHGR (SEQ ID NO: 2). Other preferredmpl ligand fragments include those produced as a result of chemical orenzymatic digestion of the purified ligand.

Another preferred aspect of the invention, is a method for purifying mplligand molecules comprises contacting a mpl ligand source containing thempl ligand molecules to be purified with an immobilized receptorpolypeptide, specifically mpl or a mpl fusion polypeptide, underconditions whereby the mpl ligand molecules to be purified areselectively adsorbed onto the immobilized receptor polypeptide, washingthe immobilized support to remove non-adsorbed material, and eluting themolecules to be purified from the immobilized receptor polypeptide towhich they are adsorbed with an elution buffer. Preferably the sourcecontaining the mpl ligand is plasma, more preferably aplastic porcineplasma and the immobilized receptor is preferably a mpl-IgG fusion. Alsopreferably the immobilized support is washed with PBS/PBS in 2M NaCl andthe elution buffer is 0.1M glycine-HCl, pH 2.25.

In another preferred embodiment, this invention provides an isolatedantibody capable of binding to the mpl ligand. Preferred mpl ligandisolated antibody is one that binds to mpl ligand with an affinity of atleast about 10⁶ l/mole. More preferably the antibody binds with anaffinity of at least about 10⁷ l/mole. Most preferably, the antibody israised against the mpl ligand having one of the above described effectorfunctions. The isolated antibody capable of binding to the mpl ligandmay optionally be fused to a second polypeptide and the antibody orfusion thereof may be used to isolate and purify mpl ligand from asource as described above for immobilized mpl polypeptide. In a furtherpreferred aspect of this embodiment, the invention provides a method fordetecting the mpl ligand in vitro or in vivo comprising contacting theantibody with a sample, especially a serum sample, suspected ofcontaining the ligand and detecting if binding has occurred.

In still further preferred embodiments, the invention provides anisolated nucleic acid molecule, encoding the mpl ligand or fragmentsthereof, which nucleic acid molecule may be labeled or unlabled with adetectable moiety, and a nucleic acid molecule having a sequence that iscomplementary to, or hybridizes under stringent or moderately stringentconditions with, a nucleic acid molecule having a sequence encoding ampl ligand. Moderately stringent conditions are conditions such asovernight incubation at 37° C. in a solution comprising: 20% formamide,5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt's solution, 10% dextrane sulfate, and 20 μl/mldenatured sheared salmon sperm DNA, followed by washing the filters in1×SSC at about 50° C. A preferred mpl ligand nucleic acid is RNA or DNAthat encodes a biologically active mpl ligand sharing at least 75%sequence identity, more preferably at least 80%, still more preferablyat least 85%, even more preferably 90%, and most preferably 95% sequenceidentity with the porcine mpl ligand.

In a further preferred aspect of this embodiment, the nucleic acidmolecule is DNA encoding the mpl ligand and further comprises areplicable vector in which the DNA is operably linked to controlsequences recognized by a host transformed with the vector. This aspectfurther includes host cells transformed with the vector and a method ofusing the DNA to effect production of mpl ligand, comprising expressingthe DNA encoding the mpl ligand in a culture of the transformed hostcells and recovering the mpl ligand from the host cell culture. The mplligand prepared in this manner is preferably human mpl ligand.

The invention further includes a preferred method for treating a mammalhaving thrombocytopenia comprising administering a therapeuticallyeffective amount of a mpl Ligand to the mammal. Preferably the mplLigand is administered in combination with a cytokine, especially acolony stimulating factor or interleukin. Preferred colony stimulatingfactors or interleukins include; G-CSF, CSF-1, GM-CSF, M-CSF,erythropoietin, IL-1, IL-3, IL-4, IL-5, IL-6, IL-7, or IL-8.

III. Methods of Making

1. Purification and Identification of mpl Ligand from Plasma

Aplastic plasma from a variety of species is known to contain activitiesthat stimulate hematopoiesis in-vitro; however no hematopoieticstimulatory factor has previously been reported isolated from plasma.One source of aplastic plasma is that obtained from irradiated pigs.This aplastic porcine plasma (APP) stimulates human hematopoiesisin-vitro. To determine if APP contained the mpl ligand, its effect on³H-thymidine incorporation into Ba/F3 cells transfected with human mpl P(Ba/F3-mpl) was measured. APP stimulated ³H-thymidine incorporation intoBa/F3-mpl cells but not Ba/F3 control cells (i.e. not transfected withhuman mpl P). No such activity was observed in normal porcine plasma.These results indicated that APP contained a factor(s) that transduces aproliferative signal through the mpl receptor and therefore may be thenatural ligand for this receptor. This was further supported by thefinding that soluble mpl-IgG blocked the stimulatory effects of APP onBa/F3-mpl cells.

The activity in APP appeared to be a protein since pronase, DTT, or heatdestroy the activity in APP (FIG. 1). The activity was alsonon-dialyzable. The activity was, however, stable to low pH (pH 2.5 for2 hrs. ) and was shown to bind and elute from several lectin-affinitycolumns, indicating that it was a glycoprotein. To further elucidate thestructure and identity of this activity it was affinity purified fromAPP.

5 liters of APP was purified according to the protocol in Example I. Therecovery of activity from each step is shown in FIG. 2 and foldpurification is provided in Table 1. The overall recovery of activitythrough the mpl-affinity column was approximately 10%. The peak activityfraction (F6) from the mpl-affinity column has an estimated specificactivity of 9.8×10⁶ units/mg. The overall purification from 5 L of APPwas approximately 4×10⁶ fold (0.8 units/mg to 3.3×10⁶ units/mg) with a83×10⁶ fold reduction in protein (250 gms to 3 μg );

TABLE 1 Purification of mpl ligand Specific Volume Protein ActivityYield Fold Sample mls mg/ml Units/ml Units Units/mg % Purification APP5000 50 40 200,000 0.8 — 1 Phenyl 4700 0.8 40 200,000 50 94 62 Blue-Sep.640 0.93 400 256,000 430 128 538 mpl-UL 12 5 × 10⁻⁴ 1666 20,0003,300,000 10 4,100,000 (Fxns 5-7)

Protein was determined by the Bradford assay. Protein concentration ofmpl-eluted fractions 5-7 are estimates based on staining intensity of asilver stained SDS-gel. One unit is defined as that causing 50% maximalstimulation of Ba/F3-mpl cell proliferation.

Analysis of eluted fractions from the mpl-affinity column by SDS-PAGE(4-20%, novex gel) run under reducing conditions, reveal the presence ofseveral proteins (FIG. 3). Proteins that silver stain with the strongestintensity resolve with apparent Mr of 66,000, 55,000, 30,000, 28,000 and14,000. To determine which of these proteins stimulate proliferationBa/F3-mpl cell cultures these proteins were eluted from the gel asdescribed Example II.

The results of this experiment show that most of the activity elutesfrom a gel slice that includes proteins with Mr 25,000-35,000, withlesser activity eluting in the 22,000-24,000 region of the gel (FIG. 4).

To identify and obtain protein sequence for the proteins resolving inthis region of the gel, fraction 6 was electroblotted and sequenced asdescribed in Example III. The only proteins that .were visable in the Mr22,000-35,000 region of the blot had Mr of 30,000, 28,000 and 22,000.

Bands at 30, 28 and 22 KDa were subjected to protein sequencing asdescribed in Example IV. Protein sequences obtained were as follows:

1) 30 KDa   1       5        10         15         20             25 (S)P A P P A(C)D P R L L N K L L R D D (H/S) V L H (G) R L (SEQ ID NO.: 3)2) 28 KDa   1        5        10        15         20               25(S) P A P P A X D P R L L N K L L R D D (H)     V L (H) G R (SEQ ID NO.:4) 3) 22 KDa 1       5         10 X P A P P A X D P R L X (N) (K) (SEQID NO.: 5)

Computer-assisted analysis revealed that these sequences were novel .The fact that these were the only sequences obtained indicates that the30 KDa, 28 KDa and 22 kDa proteins are related and may be differentforms of the same novel protein. Futhermore this protein(s) is a likelycandidate as the natural mpl-ligand since the activity resolved onSDS-PAGE in the same region of a 4-20% gel (22,000-35,000). Futhermorethe partially purified ligand migrates with a Mr of 17,000-30,000 whensubjected to gel filtration chromatography using a Superose 12(Pharmacia) column. The different Mr forms of the ligand are likely aresult of proteolysis or glycosylation differences or other post orpre-translational modifications.

As described earlier, antisense human mpl RNA abrogatedmegakaryocytopoiesis in human bone marrow cultures enriched with CD 34+progenitor cells without affecting the differentiation of otherhemopoietic cell lineages (Methia, N et al., supra). This resultsuggested that the mpl receptor plays a role in the differentiation andproliferation of megakaryocytes in-vitro. To further elucidate the roleof the mpl-ligand in megakaryocytopoiesis, the effects of APP andmpl-ligand depleted APP on in-vitro human megakaryocytopoiesis wascompared. The effect of APP on human megakaryocytopoiesis was determinedusing a modification of the liquid suspension megakaryocytopoiesis assaydescribed by Solberg et al. (Example IV). In this assay human peripheralstem cells (PSC) are treated with APP before and after mpl-IgG affinitychromatography. GP II_(b)III_(a) stimulation or megakaryocytopoiesis isquantitated with an ¹²⁵I anti-II_(b)III_(a) antibody (FIG. 5). Shown inFIG. 5 10% APP caused approximately a 3 fold stimulation while APPdepleted of mpl-ligand had no effect. Significantly, the mpl-liganddepleted APP did not induce proliferation of the Ba/F3-mpl cells.

In another experiment, soluble human mpl-IgG added at days 0, 2 and 4 tocultures containing 10% APP neutralized the stimulatory effects of APPon human megakaryocytopoiesis (FIG. 6). These results indicate that thempl-ligand plays a role in regulating human megakaryocytopoiesis andtherefore may be useful for the treatment of thrombocytopenia.

2. Additional Methods for Measurement of Thrombopoeitic activity

In addition to the methods described immediately above, thrombopoeiticactivity may be measured in various assays including an in-vivo mouseplatelet rebound synthesis assay, induction of platelet cell surfaceantigen assay as measured by an anti-platelet immunoassay(anti-GPII_(b)III_(a)) for a human leukaemia megakaryoblastic cell line(CMK)(see Sato, T., et al., Brit. J. Heamatol. 72:184-190 (1989)), andinduction of polyploidization in a megakaryoblastic cell line(DAMI) (seeOgura, M., et al., Blood 72(1):49-60 (1988). Maturation ofmegakaryocytes from immature, largely non-DNA synthesizing cells, tomorphologically identifiable megakaryocytes involves a process thatincludes appearance of cytoplasmic organelles, acquisition of membraneantigens (GPII_(b)III_(a)), endoreplication and release of platelets asdescribed in the background. A lineage specific promoter (i.e. the mplligand) of megakaryocyte maturation, would be expected to induce atleast some of these changes in immature megakaryocytes leading toplatelet release and alleviation of thympocytopenia. Thus, assays weredesigned to measure the emergence of these parameters in immaturemegakaryocyte cell lines, i.e. CMK and DAMI cells. The CMK assay(Example VI) measures the appearance of a specific platelet marker,GPII_(b)III_(a), and platelet shedding. The DAMI assay (Example VII)measures endoreplication since increases in ploidy are hallmarks ofmature megakaryocytes. Recognizable megakaryocytes have ploidy values of2N, 4N, 8N, 16N, 32N, etc. Finally, the in vivo assay (Example VIII) areuseful in demonstrating that administration of the test compound (herethe mpl ligand) results in elevation of platelet numbers.

3. Recombinant Preparation of Mpl Ligand

Preferably mpl ligand is prepared by standard recombinant procedureswhich involves production of the mpl ligand polypeptide by culturingcells transfected to express mpl ligand nucleic acid (typically bytransforming the cells with an expression vector) and recovering thepolypeptide from the cells. However, it is optionally envisioned thatthe mpl ligand may be produced by homologous recombination, or withrecombinant production methods utilizing control elements introducedinto cells already containing DNA encoding the mpl ligand. For example,a powerful promoter/enhancer element, a suppressor, or an exogenoustranscription modulatory element may be inserted in the genome of theintended host cell in proximity and orientation sufficient to influencethe transcription of DNA encoding the desired mpl ligand polypeptide.The control element does not encode the mpl ligand, rather the DNA isindigenous to the host cell genome. One next screens for cells makingthe receptor polypeptide of this invention, or for increased ordecreased levels of expression, as desired.

Thus, the invention contemplates a method for producing mpl ligandcomprising inserting into the genome of a cell containing the mpl ligandnucleic acid molecule a transcription modulatory element in sufficientproximity and orientation to the nucleic acid molecule to influencetranscription thereof, with an optional further step comprisingculturing the cell containing the transcription modulatory element andthe nucleic acid molecule. The invention also contemplates a host cellcontaining the indigenous mpl ligand nucleic acid molecule operablylinked to exogenous control sequences recognized by the host cell.

A. Isolation of DNA Encoding mpl Ligand Polypeptide

The DNA encoding mpl ligand polypeptide may be obtained from any cDNAlibrary prepared from tissue believed to possess the mpl ligand mRNA andto express it at a detectable level. The mpl ligand gene may also beobtained from a genomic DNA library or by in vitro oligonucleotidesynthesis from the complete nucleotide or amino acid sequence.

Libraries are screened with probes designed to identify the gene ofinterest or the protein encoded by it. For cDNA expression libraries,suitable probes include monoclonal or polyclonal antibodies thatrecognize and specifically bind to the mpl ligand. For cDNA librariessuitable probes include oligonucleotides of about 20-80 bases in lengththat encode known or suspected portions of the mpl ligand cDNA from thesame or different species; and/or complementary or homologous cDNAs orfragments thereof that encode the same or a similar gene. Appropriateprobes for screening genomic DNA libraries include, but are not limitedto, oligonucleotides, cDNAs, or fragments thereof that encode the sameor a similar gene, and/or homologous genomic DNAs or fragments thereof.Screening the cDNA or genomic library with the selected probe may beconducted using standard procedures as described in chapters 10-12 ofSambrook et al., supra.

An alternative means to isolate the gene encoding mpl ligand is to usePCR methodology as described in section 14 of Sambrook et al., supra.This method requires the use of oligonucleotide probes that willhybridize to DNA encoding the mpl ligand. Strategies for selection ofoligonucleotides are described below.

A preferred method of practicing this invention is to use carefullyselected oligonucleotide sequences to screen cDNA libraries from varioustissues, preferably porcine kidney cell lines. Optionally, human kidneycell line cDNA libraries are screened with the oligonucleotide probes.Alternatively, human genomic libraries may be screened with theoligonucleotide probes.

The oligonucleotide sequences selected as probes should be of sufficientlength and sufficiently unambiguous that false positives are minimized.The actual nucleotide sequence(s) is usually designed based on regionsof the mpl ligand which have the least codon redundancy. Theoligonucleotides may be degenerate at one or more positions. The use ofdegenerate oligonucleotides is of particular importance where a libraryis screened from a species in which preferential codon usage is notknown.

The oligonucleotide must be labeled such that it can be detected uponhybridization to DNA in the library being screened. The preferred methodof labeling is to use ATP and polynucleotide kinase to radiolabel the 5′end of the oligonucleotide. However, other methods may be used to labelthe oligonucleotide, including, but not limited to, biotinylation orenzyme labeling.

Of particular interest is the mpl ligand nucleic acid that encodes afull-length mpl ligand polypeptide. In some preferred embodiments, thenucleic acid sequence includes the native mpl ligand signal sequence.Nucleic acid having all the protein coding sequence is obtained byscreening selected cDNA or genomic libraries using the deduced aminoacid sequence.

B. Amino Acid Sequence Variants of Native mpl Ligand

Amino acid sequence variants of mpl ligand are prepared by introducingappropriate nucleotide changes into the mpl ligand DNA, or by in vitrosynthesis of the desired mpl ligand polypeptide. Such variants include,for example, deletions from, or insertions or substitutions of, residueswithin the amino acid sequence for the porcine mpl ligand. Anycombination of deletion, insertion, and substitution is made to arriveat the final construct, provided that the final construct possesses thedesired characteristics. The amino acid changes also may alterpost-translational processes of the mpl ligand, such as changing thenumber or position of glycosylation sites.

For the design of amino acid sequence variants of the mpl ligand, thelocation of the mutation site and the nature of the mutation will dependon the mpl ligand characteristic(s) to be modified. The sites formutation can be modified individually or in series, e.g., by (1)substituting first with conservative amino acid choices and then withmore radical selections depending upon the results achieved, (2)deleting the target residue, or (3) inserting residues of the same or adifferent class adjacent to the located site, or combinations of options1-3.

A useful method for identification of certain residues or regions of thempl ligand polypeptide that are preferred locations for mutagenesis iscalled “alanine scanning mutagenesis,” as described by Cunningham andWells, Science, 244:1081-1085 (1989). Here, a residue or group of targetresidues are identified (e.g., charged residues such as arg, asp, his,lys, and glu) and replaced by any but preferably a neutral or negativelycharged amino acid (most preferably alanine or polyalanine) to affectthe interaction of the amino acids with the surrounding aqueousenvironment in or outside the cell. Those domains demonstratingfunctional sensitivity to the substitutions then are refined byintroducing further or other variants at or for the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to optimize the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressed mpl ligandvariants are screened for the optimal combination of desired activity.

There are two principal variables in the construction of amino acidsequence variants: the location of the mutation site and the nature ofthe mutation. For example, variants of the mpl ligand polypeptideinclude variants from the mpl ligand sequence, and may representnaturally occurring alleles (which will not require manipulation of thempl ligand DNA) or predetermined mutant. forms made by mutating the DNA,either to arrive at an allele or a variant not found in nature. Ingeneral, the location and nature of the mutation chosen will depend uponthe mpl ligand characteristic to be modified.

Amino acid sequence deletions generally range from about 1 to 30residues, more preferably, about 1 to 10 residues, and typically arecontiguous. Contiguous deletions ordinarily are made in even numbers ofresidues, but single or odd numbers of deletions are within the scopehereof. Deletions may be introduced into regions of low homology amongthe mpl ligands that share the most sequence identity to modify theactivity of the mpl ligand. Or deletions may be introduced into regionsof low homology among human mpl ligand and other mamalian mpl ligandpolypeptides that share the most sequence identity to the human mplligand. Deletions from a mammalian mpl ligand polypeptide in areas ofsubstantial homology with other mammalian mpl ligands will be morelikely to modify the biological activity of the mpl ligand moresignificantly. The number of consecutive deletions will be selected soas to preserve the tertiary structure of mpl ligands in the affecteddomain, e.g., beta-pleated sheet or alpha helix.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Intrasequence insertions (i.e.,insertions within the mature mpl ligand sequence) may range generallyfrom about 1 to 10 residues, more preferably 1 to 5, most preferably 1to 3. An exemplary preferred fusion is that of mpl ligand or fragmentthereof and another cytokine or fragment thereof. Insertions are oftenmade in even numbers of residues, but this is not required. Examples ofterminal insertions include mature mpl ligand with an N-terminalmethionyl residue, an artifact of the direct expression of mature mplligand in recombinant cell culture, and fusion of a heterologousN-terminal signal sequence to the N-terminus of the mature mpl ligandmolecule to facilitate the secretion of mature mpl ligand fromrecombinant hosts. Such signal sequences generally will be obtainedfrom, and thus homologous to, the intended host cell species. Suitablesequences include STII or Ipp for E. coli, alpha factor for yeast, andviral signals such as herpes gD for mammalian cells.

Other insertional variants of the mpl ligand molecule include the fusionto the N- or C-terminus of mpl ligand of immunogenic polypeptides (i.e.,not endogenous to the host to which the fusion is administered), e.g.,bacterial polypeptides such as beta-lactamase or an enzyme encoded bythe E. coli trp locus, or yeast protein, and C-terminal fusions withproteins having a long half-life such as immunoglobulin constant regions(or other immunoglobulin regions), albumin, or ferritin, as described inWO 89/02922 published Apr. 6, 1989.

A third group of variants are amino acid substitution variants. Thesevariants have at least one amino acid residue in the mpl ligand moleculeremoved and a different residue inserted in its place. The sites ofgreatest interest for substitutional mutagenesis include sitesidentified as the active site(s) of mpl ligand and sites where the aminoacids found in other analogues are substantially different in terms ofside-chain bulk, charge, or hydrophobicity, but where there is also ahigh degree of sequence identity at the selected site among various mplligand species and/or within the various animal analogues of one mplligand member.

Other sites of interest are those in which particular residues of thempl ligand obtained from various family members and/or animal specieswithin one member are identical. These sites, especially those fallingwithin a sequence of at least three other identically conserved sites,are substituted in a relatively conservative manner. Such conservativesubstitutions are shown in Table 2 under the heading of preferredsubstitutions. If such substitutions result in a change in biologicalactivity, then more substantial changes, denominated exemplarysubstitutions in Table 2, or as further described below in reference toamino acid classes, are introduced and the products screened.

TABLE 2 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his;lys; arg gln Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) aspasp Gly (G) pro pro His (H) asn; gln; lys; arg arg Ile (I) leu; val;met; ala; phe; leu norleucine Leu (L) norleucine; ile; val; ile met;ala; phe Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F)leu; val; ile; ala leu Pro (P) gly gly Ser (S) thr thr Thr (T) ser serTrp (W) tyr tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met;phe; leu ala; norleucine

Substantial modifications in function or immunological identity of thempl ligand are accomplished by selecting substitutions that differsignificantly in their effect on maintaining (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside-chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gln, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another. Such substituted residues also may beintroduced into the conservative substitution sites or, more preferably,into the remaining (non-conserved) sites.

In one embodiment of the invention, it is desirable to inactivate one ormore protease cleavage sites that are present in the molecule. Thesesites are identified by inspection of the encoded amino acid sequence,in the case of trypsin, e.g., for an arginyl or lysinyl residue. Whenprotease cleavage sites are identified, they are rendered inactive toproteolytic cleavage by substituting the targeted residue with anotherresidue, preferably a basic residue such as glutamine or a hydrophobicresidue such as serine; by deleting the residue; or by inserting aprolyl residue immediately after the residue.

In another embodiment, any methionyl residues other than the startingmethionyl residue of the signal sequence, or any residue located withinabout three residues N- or C-terminal to each such methionyl residue, issubstituted by another residue (preferably in accord with Table 2) ordeleted. Alternatively, about 1-3 residues are inserted adjacent to suchsites.

Any cysteine residues not involved in maintaining the properconformation of the mpl ligand also may be substituted, generally withserine, to improve the oxidative stability of the molecule and preventaberrant crosslinking.

Nucleic acid molecules encoding amino acid sequence variants of mplligand are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence variants) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared variantor a non-variant version of mpl ligand polypeptide.

Oligonucleotide-mediated mutagenesis is a preferred method for preparingsubstitution, deletion, and insertion variants of mpl ligand DNA. Thistechnique is well known in the art as described by Adelman et al., DNA,2:183 (1983). Briefly, mpl ligand DNA is altered by hybridizing anoligonucleotide encoding the desired mutation to a DNA template, wherethe template is the single-stranded form of a plasmid or bacteriophagecontaining the unaltered or native DNA sequence of mpl ligand. Afterhybridization, a DNA polymerase is used to synthesize an entire secondcomplementary strand of the template that will thus incorporate theoligonucleotide primer, and will code for the selected alteration in thempl ligand DNA.

Generally, oligonucleotides of at least 25 nucleotides in length areused. An optimal oligonucleotide will have 12 to 15 nucleotides that arecompletely complementary to the template on either side of thenucleotide(s) coding for the mutation. This ensures that theoligonucleotide will hybridize properly to the single-stranded DNAtemplate molecule. The oligonucleotides are readily synthesized usingtechniques known in the art such as that described by Crea et al., Proc.Nat. Acad. Sci. USA, 75:5765 (1978).

The DNA template can be generated by those vectors that are eitherderived from bacteriophage M13 vectors (the commercially available M13mp 18 and M13 mp 19 vectors are suitable), or those vectors that containa single-stranded phage origin of replication as described by Viera etal. Meth. Enzymol., 153:3 (1987). Thus, the DNA that is to be mutatedmay be inserted into one of these vectors to generate single-strandedtemplate. Production of the single-stranded template is described inSections 4.21-4.41 of Sambrook et al., Molecular Cloning: A LaboratoryManual (Cold Spring Harbor Laboratory Press, NY 1989).

Alternatively, single-stranded DNA template may be generated bydenaturing double-stranded plasmid (or other) DNA using standardtechniques.

For alteration of the native DNA sequence (to generate amino acidsequence variants, for example), the oligonucleotide is hybridized tothe single-stranded template under suitable hybridization conditions. ADNA polymerizing enzyme, usually the Klenow fragment of DNA polymeraseI, is then added to synthesize the complementary strand of the templateusing the oligonucleotide as a primer for synthesis. A heteroduplexmolecule is thus formed such that one strand of DNA encodes the mutatedform of the mpl ligand, and the other strand (the original template)encodes the native, unaltered sequence of the mpl ligand. Thisheteroduplex molecule is then transformed into a suitable host cell,usually a prokaryote such as E. coli JM101. After the cells are grown,they are plated onto agarose plates and screened using theoligonucleotide primer radiolabeled with 32-phosphate to identify thebacterial colonies that contain the mutated DNA. The mutated region isthen removed and placed in an appropriate vector for protein production,generally an expression vector of the type typically employed fortransformation of an appropriate host.

The method described immediately above may be modified such that ahomoduplex molecule is created wherein both strands of the plasmidcontain the mutation(s). The modifications are as follows: Thesingle-stranded oligonucleotide is annealed to the single-strandedtemplate as described above. A mixture of three deoxyribonucleotides,deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP), anddeoxyribothymidine (dTTP), is combined with a modifiedthio-deoxyribocytosine called dCTP-(aS) (which can be obtained from theAmersham Corporation). This mixture is added to thetemplate-oligonucleotide complex. Upon addition of DNA polymerase tothis mixture, a strand of DNA identical to the template except for themutated bases is generated. In addition, this new strand of DNA willcontain dCTP-(aS) instead of dCTP, which serves to protect it fromrestriction endonuclease digestion.

After the template strand of the double-stranded heteroduplex is nickedwith an appropriate restriction enzyme, the template strand can bedigested with ExoIII nuclease or another appropriate nuclease past theregion that contains the site(s) to be mutagenized. The reaction is thenstopped to leave a molecule that is only partially single-stranded. Acomplete double-stranded DNA homoduplex is then formed using DNApolymerase in the presence of all four deoxyribonucleotidetriphosphates, ATP, and DNA ligase. This homoduplex molecule can then betransformed into a suitable host cell such as E. coli JM101, asdescribed above.

DNA encoding mpl ligand mutants with more than one amino acid to besubstituted may be generated in one of several ways. If the amino acidsare located close together in the polypeptide chain, they may be mutatedsimultaneously using one oligonucleotide that codes for all of thedesired amino acid substitutions. If, however, the amino acids arelocated some distance from each other (separated by more than about tenamino acids), it is more difficult to generate a single oligonucleotidethat encodes all of the desired changes. Instead, one of two alternativemethods may be employed.

In the first method, a separate oligonucleotide is generated for eachamino acid to be substituted. The oligonucleotides are then annealed tothe single-stranded template DNA simultaneously, and the second strandof DNA that is synthesized from the template will encode all of thedesired amino acid substitutions.

The alternative method involves two or more rounds of mutagenesis toproduce the desired mutant. The first round is as described for thesingle mutants: wild-type DNA is used for the template, anoligonucleotide encoding the first desired amino acid substitution(s) isannealed to this template, and the heteroduplex DNA molecule is thengenerated. The second round of mutagenesis utilizes the mutated DNAproduced in the first round of mutagenesis as the template. Thus, thistemplate already contains one or more mutations. The oligonucleotideencoding the additional desired amino acid substitution(s) is thenannealed to this template, and the resulting strand of DNA now encodesmutations from both the first and second rounds of mutagenesis. Thisresultant DNA can be used as a template in a third round of mutagenesis,and so on. PCR mutagenesis is also suitable for making amino acidvariants of mpl ligand polypeptide. While the following discussionrefers to DNA, it is understood that the technique also findsapplication with RNA. The PCR technique generally refers to thefollowing procedure (see Erlich, supra, the chapter by R. Higuchi, p.61-70): When small amounts of template DNA are used as starting materialin a PCR, primers that differ slightly in sequence from thecorresponding region in a template DNA can be used to generaterelatively large quantities of a specific DNA fragment that differs fromthe template sequence only at the positions where the primers differfrom the template. For introduction of a mutation into a plasmid DNA,one of the primers is designed to overlap the position of the mutationand to contain the mutation; the sequence of the other primer must beidentical to a stretch of sequence of the opposite strand of theplasmid, but this sequence can be located anywhere along the plasmidDNA. It is preferred, however, that the sequence of the second primer islocated within 200 nucleotides from that of the first, such that in theend the entire amplified region of DNA bounded by the primers can beeasily sequenced. PCR amplification using a primer pair like the onejust described results in a population of DNA fragments that differ atthe position of the mutation specified by the primer, and possibly atother positions, as template copying is somewhat error-prone.

If the ratio of template to product material is extremely low, the vastmajority of product DNA fragments incorporate the desired mutation(s).This product material is used to replace the corresponding region in theplasmid that served as PCR template using standard DNA technology.Mutations at separate positions can be introduced simultaneously byeither using a mutant second primer, or performing a second PCR withdifferent mutant primers and ligating the two resulting PCR fragmentssimultaneously to the vector fragment in a three (or more)-partligation.

In a specific example of PCR mutagenesis, template plasmid DNA (1 μg) islinearized by digestion with a restriction endonuclease that has aunique recognition site in the plasmid DNA outside of the region to beamplified. Of this material, 100 ng is added to a PCR mixture containingPCR buffer, which contains the four deoxynucleotide triphosphates and isincluded in the GeneAmp® kits (obtained from Perkin-Elmer Cetus,Norwalk, Conn. and Emeryville, Calif.), and 25 pmole of eacholigonucleotide primer, to a final volume of 50 μl. The reaction mixtureis overlayed with 35 μl mineral oil. The reaction mixture is denaturedfor five minutes at 100° C., placed briefly on ice, and then 1 μlThermus aquaticus (Taq) DNA polymerase (5 units/μl, purchased fromPerkin-Elmer Cetus) is added below the mineral oil layer. The reactionmixture is then inserted into a DNA Thermal Cycler (purchased fromPerkin-Elmer Cetus) programmed as follows:

2 min. 55° C.

30 sec. 72° C., then 19 cycles of the following:

30 sec. 94° C.

30 sec. 55° C., and

30 sec. 72° C.

At the end of the program, the reaction vial is removed from the thermalcycler and the aqueous phase transferred to a new vial, extracted withphenol/chloroform (50:50 vol), and ethanol precipitated, and the DNA isrecovered by standard procedures. This material is subsequentlysubjected to the appropriate treatments for insertion into a vector.

Another method for preparing variants, cassette mutagenesis, is based onthe technique described by Wells, et al., Gene, 34:315 (1985). Thestarting material is the plasmid (or other vector) comprising the mplligand DNA to be mutated. The codon(s) in the mpl ligand DNA to bemutated are identified. There must be a unique restriction endonucleasesite on each side of the identified mutation site(s). If no suchrestriction sites exist, they may be generated using the above-describedoligonucleotide-mediated mutagenesis method to introduce them atappropriate locations in the mpl ligand DNA. After the restriction siteshave been introduced into the plasmid, the plasmid is cut at these sitesto linearize it. A double-stranded oligonucleotide encoding the sequenceof the DNA between the restriction sites but containing the desiredmutation(s) is synthesized using standard procedures. The two strandsare synthesized separately and then hybridized together using standardtechniques. This double-stranded oligonucleotide is referred to as thecassette. This cassette is designed to have 3′ and 5′ ends that arecompatible with the ends of the linearized plasmid, such that it can bedirectly ligated to the plasmid. This plasmid now contains the mutatedmpl ligand DNA sequence.

C. Insertion of Nucleic Acid into a Replicable Vector

The nucleic acid (e.g., cDNA or genomic DNA) encoding native or variantmpl ligand polypeptide is inserted into a replicable vector for furthercloning (amplification of the DNA) or for expression. Many vectors areavailable, and selection of the appropriate vector will depend on 1)whether it is to be used for DNA amplification or for DNA expression, 2)the size of the nucleic acid to be inserted into the vector, and 3) thehost cell to be transformed with the vector. Each vector containsvarious components depending on its function (amplification of DNA orexpression of DNA) and the host cell with which it is compatible. Thevector components generally include, but are not limited to, one or moreof the following: a signal sequence, an origin of replication, one ormore marker genes, an enhancer element, a promoter, and a transcriptiontermination sequence.

(i) Signal Sequence Component

The mpl ligand of this invention may be expressed not only directly, butalso as a fusion with a heterologous polypeptide, preferably a signalsequence or other polypeptide having a specific cleavage site at theN-terminus of the mature protein or polypeptide. In general, the signalsequence may be a component of the vector, or it may be a part of thempl ligand DNA that is inserted into the vector. The heterologous signalsequence selected should be one that is recognized and processed (i.e.,cleaved by a signal peptidase) by the host cell. For prokaryotic hostcells that do not recognize and process the native mpl ligand signalsequence, the signal sequence is substituted by a prokaryotic signalsequence selected, for example, from the group of the alkalinephosphatase, penicillinase, Ipp, or heat-stable enterotoxin II leaders.For yeast secretion the native signal sequence may be substituted by,e.g., the yeast invertase, alpha factor, or acid phosphatase leaders,the C. albicans glucoamylase leader (EP 362,179 published Apr. 4, 1990),or the signal described in WO 90/13646 published Nov. 15, 1990. Inmammalian cell expression the native signal sequence (i.e., the mplligand presequence that normally directs secretion of mpl ligand fromits naitve mammalian cells in vivo) is satisfactory, although othermammalian signal sequences may be suitable, such as signal sequencesfrom other mpl ligand polypeptides or from the same mpl ligand from adifferent animal species, signal sequences from a mpl ligand, and signalsequences from secreted polypeptides of the same or related species, aswell as viral secretory leaders, for example, the herpes simplex gDsignal.

(ii) Origin of Replication Component

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)are useful for cloning vectors in mammalian cells. Generally, the originof replication component is not needed for mammalian expression vectors(the SV40 origin may typically be used only because it contains theearly promoter).

Most expression vectors are “shuttle” vectors, i.e., they are capable ofreplication in at least one class of organisms but can be transfectedinto another organism for expression. For example, a vector is cloned inE. coli and then the same vector is transfected into yeast or mammaliancells for expression even though it is not capable of replicatingindependently of the host cell chromosome.

DNA may also be amplified by insertion into the host genome. This isreadily accomplished using Bacillus species as hosts, for example, byincluding in the vector a DNA sequence that is complementary to asequence found in Bacillus genomic DNA. Transfection of Bacillus withthis vector results in homologous recombination with the genome andinsertion of mpl ligand DNA. However, the recovery of genomic DNAencoding mpl ligand is more complex than that of an exogenouslyreplicated vector because restriction enzyme digestion is required toexcise the mpl ligand DNA.

(iii) Selection Gene Component

Expression and cloning vectors should contain a selection gene, alsotermed a selectable marker. This gene encodes a protein necessary forthe survival or growth of transformed host cells grown in a selectiveculture medium. Host cells not transformed with the vector containingthe selection gene will not survive in the culture medium. Typicalselection genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate,or tetracycline, (b) complement auxotrophic deficiencies, or (c) supplycritical nutrients not available from complex media, e.g., the geneencoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene express a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin Southern et al (J. Molec. Appl. Genet., 1: 327[1982]) mycophenolic acid Mulligan et al (Science, 209:1422 [1980]) orhygromycin (Sugden et al, Mol. Cell. Biol., 5: 410-413 [1985]). Thethree examples given above employ bacterial genes under eukaryoticcontrol to convey resistance to the appropriate drug G418 or neomycin(geneticin), xgpt (mycophenolic acid), or hygromycin, respectively.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up thempl ligand nucleic acid, such as dihydrofolate reductase (DHFR) orthymidine kinase. The mammalian cell transformants are placed underselection pressure that only the transformants are uniquely adapted tosurvive by virtue of having taken up the marker. Selection pressure isimposed by culturing the transformants under conditions in which theconcentration of selection agent in the medium is successively changed,thereby leading to amplification of both the selection gene and the DNAthat encodes mpl ligand polypeptide. Amplification is the process bywhich genes in greater demand for the production of a protein criticalfor growth are reiterated in tandem within the chromosomes of successivegenerations of recombinant cells. Increased quantities of mpl ligand aresynthesized from the amplified DNA.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity, prepared andpropagated as described by Urlaub and Chasin, Proc. Natl. Acad. Sci.USA, 77:4216 [1980]. The transformed cells are then exposed to increasedlevels of Mtx. This leads to the synthesis of multiple copies of theDHFR gene, and, concomitantly, multiple copies of other DNA comprisingthe expression vectors, such as the DNA encoding mpl ligand. Thisamplification technique can be used with any otherwise suitable host,e.g., ATCC No. CCL61 CHO-K1, notwithstanding the presence of endogenousDHFR if, for example. a mutant DHFR gene that is highly resistant to Mtxis employed (EP 117,060). Alternatively, host cells [particularlywild-type hosts that contain endogenous DHFR] transformed orco-transformed with DNA sequences encoding mpl ligand, wild-type DHFRprotein, and another selectable marker such as aminoglycoside 3′phosphotransferase (APH) can be selected by cell growth in mediumcontaining a selection agent for the selectable marker such as anaminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See U.S.Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 [1979];Kingsman et al., Gene, 7:141 [1979]; or Tschemper et al., Gene, 10: 157[1980]). The trp1 gene provides a selection marker for a mutant strainof yeast lacking the ability to grow in tryptophan, for example, ATCCNo. 44076 or PEP4-1 (Jones, Genetics, 85: 12 [1977]). The presence ofthe trp1 lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

(iv) Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the mpl ligandnucleic acid. Promoters are untranslated sequences located upstream (5′)to the start codon of a structural gene (generally within about 100 to1000 bp) that control the transcription and translation of particularnucleic acid sequence, such as the mpl ligand nucleic acid sequence, towhich they are operably linked. Such promoters typically fall into twoclasses, inducible and constitutive. Inducible promoters are promotersthat initiate increased levels of transcription from DNA under theircontrol in response to some change in culture conditions, e.g., thepresence or absence of a nutrient or a change in temperature. At thistime a large number of promoters recognized by a variety of potentialhost cells are well known. These promoters are operably linked to mplligand encoding DNA by removing the promoter from the source DNA byrestriction enzyme digestion and inserting the isolated promotersequence into the vector. Both the native mpl ligand promoter sequenceand many heterologous promoters may be used to direct amplificationand/or expression of the mpl ligand DNA. However, heterologous promotersare preferred, as they generally permit greater transcription and higheryields of expressed mpl ligand as compared to the native mpl ligandpromoter.

Promoters suitable for use with prokaryotic hosts include theβ-lactamase and lactose promoter systems (Chang et al., Nature, 275:615[1978]; and Goeddel et al., Nature, 281: 544 [1979]), alkalinephosphatase, a tryptophan (trp) promoter system (Goeddel, Nucleic AcidsRes., 8:4057 [1980] and EP 36,776) and hybrid promoters such as the tacpromoter (deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 [1983]).However, other known bacterial promoters are suitable. Their nucleotidesequences have been published, thereby enabling a skilled workeroperably to ligate them to DNA encoding mpl ligand (Siebenlist et al.,Cell, 20:269 [1980]) using linkers or adaptors to supply any requiredrestriction sites. Promoters for use in bacterial systems also willcontain a Shine-Dalgarno (S.D.) sequence operably linked to the DNAencoding mpl ligand polypeptide.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CXCAAT region where X may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase (Hitzeman et al., J.Biol. Chem., 255:2073 [1980]) or other glycolytic enzymes (Hess et al.,J. Adv. Enzyme Reg., 7:149 [1968]; and Holland, Biochemistry, 17:4900[1978]), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, met allothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin Hitzeman et al., EP 73,657A. Yeast enhancers also are advantageouslyused with yeast promoters.

Mpl ligand transcription from vectors in mammalian host cells iscontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus (UK 2,211,504 publishedJul. 5, 1989), adenovirus (such as Adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, a retrovirus hepatitis-Bvirus and most preferably Simian Virus 40 (SV40), from heterologousmammalian promoters, e.g., the actin promoter or an immunoglobulinpromoter, from heat-shock promoters, and from the promoter normallyassociated with the mpl ligand sequence, provided such promoters arecompatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. Fiers et al., Nature, 273:113 (1978); Mulligan and Berg,Science, 209:1422-1427 (1980); Pavlakis et al., Proc. Natl. Acad. Sci.USA, 78:7398-7402 (1981). The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. Greenaway et al., Gene, 18:355-360 (1982). A system forexpressing DNA in mammalian hosts using the bovine papilloma virus as avector is disclosed in U.S. Pat. No. 4,419,446. A modification of thissystem is described in U.S. Pat. No. 4,601,978. See also Gray et al.,Nature, 295:503-508 (1982) on expressing cDNA encoding immune interferonin monkey cells; Reyes et al., Nature, 297:598-601 (1982) on expressionof human β-interferon cDNA in mouse cells under the control of athymidine kinase promoter from herpes simplex virus; Canaani and Berg,Proc. Natl. Acad. Sci. USA, 79: 5166-5170 (1982) on expression of thehuman interferon β1 gene in cultured mouse and rabbit cells; and Gormanet al., Proc. Natl. Acad. Sci. USA, 79: 6777-6781 (1982) on expressionof bacterial CAT sequences in CV-1 monkey kidney cells, chicken embryofibroblasts, Chinese hamster ovary cells, HeLa cells, and mouse NIH-3T3cells using the Rous sarcoma virus long terminal repeat as a promoter.

(v) Enhancer Element Component

Transcription of a DNA encoding the mpl ligand of this invention byhigher eukaryotes is often increased by inserting an enhancer sequenceinto the vector. Enhancers are cis-acting elements of DNA, usually aboutfrom 10 to 300 bp, that act on a promoter to increase its transcription.Enhancers are relatively orientation and position independent, havingbeen found 5′ (Laimins et al. Proc. Natl. Acad. Sci. USA, 78:993 [1981])and 3′ (Lusky et al. Mol. Cell Bio., 3:1108 [1983]) to the transcriptionunit, within an intron (Banerji et al. Cell, 33:729 [1983]), as well aswithin the coding sequence itself (Osborne et al. Mol. Cell Bio., 4:1293[1984]). Many enhancer sequences are now known from mammalian genes(globin, elastase, albumin, a-fetoprotein, and insulin). Typically,however, one will use an enhancer from a eukaryotic cellvirus. Examplesinclude the SV40 enhancer on the late side of the replication origin (bp100-270), the cytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, and adenovirusenhancers. See also Yaniv, Nature, 297:17-18 (1982) on enhancingelements for activation of eukaryotic promoters. The enhancer may bespliced into the vector at a position 5′ or 3′ to the mpl ligandencoding sequence, but is preferably located at a site 5′ from thepromoter.

(vi) Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′ untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding mpl ligand.

(vii) Construction and Analysis of Vectors

Construction of suitable vectors containing one or more of the abovelisted components employs standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and religated in theform desired to generate the plasmids required.

For analysis to confirm correct sequences in plasmids constructed, theligation mixtures are used to transform E. coli K12 strain 294 (ATCC31,446) and successful transformants selected by ampicillin ortetracycline resistance where appropriate. Plasmids from thetransformants are prepared, analyzed by restriction endonucleasedigestion, and/or sequenced by the method of Messing et al., NucleicAcids Res., 9:309 (1981) or by the method of Maxam et al., Methods inEnzymology, 65:499 (1980).

(viii) Transient Expression Vectors

Particularly useful in the practice of this invention are expressionvectors that provide for the transient expression in mammalian cells ofDNA encoding the mpl ligand polypeptide. In general, transientexpression involves the use of an expression vector that is able toreplicate efficiently in a host cell, such that the host cellaccumulates many copies of the expression vector and, in turn,synthesizes high levels of a desired polypeptide encoded by theexpression vector. Sambrook et al., supra, pp. 16.17-16.22. Transientexpression systems, comprising a suitable expression vector and a hostcell, allow for the convenient positive identification of polypeptidesencoded by cloned DNAs, as well as for the rapid screening of suchpolypeptides for desired biological or physiological properties. Thus,transient expression systems are particularly useful in the inventionfor purposes of identifying analogs and variants of mpl ligandpolypeptide that have mpl ligand polypeptide biological activity.

(ix) Suitable Exemplary Vertebrate Cell Vectors

Other methods, vectors, and host cells suitable for adaptation to thesynthesis of mpl ligand in recombinant vertebrate cell culture aredescribed in Gething et al., Nature, 293: 620-625 [1981]; Mantei et al.,Nature, 281:40-46 [1979]; Levinson et al.; EP 117,060; and EP 117,058. Aparticularly useful plasmid for mammalian cell culture expression of mplligand is pRK5 (EP pub. no. 307,247) or pSVI6B (U.S. Ser. No. 3007/441,574 filed Nov. 22, 1989, the disclosure of which is incorporatedherein by reference).

D. Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the vectors here in arethe prokaryote, yeast, or higher eukaryotic cells described above.Suitable prokaryotes include eubacteria, such as Gram-negative orGram-positive organisms, for example, E. coli, Bacilli such as B.subtilis, Pseudomonas species such as P. aeruginosa, Salmonellatyphimurium, or Serratia marcescans. One preferred E. coli cloning hostis E. coli 294 (ATCC 31,446), although other strains such as E. coli B,E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) aresuitable. These examples are illustrative rather than limiting.Preferably the host cell should secrete minimal amounts of proteolyticenzymes. Alternatively, in vitro methods of cloning, e.g., PCR or othernucleic acid polymerase reactions, are suitable.

In addition to prokaryptes, eukaryotic microbes such as filamentousfungi or yeast are suitable hosts for mpl ligand encoding vectors.Saccharomyces cerevisiae, or common baker's yeast, is the most commonlyused among lower eukaryotic host microorganisms. However, a number ofother genera, species, and strains are commonly available and usefulherein, such as Schizosaccharomyces pombe [Beach and Nurse, Nature,290:140 (1981); EP 139,383 published May 2, 1985], Kluyveromyces hosts(U.S. Pat. No. 4,943,529) such as, e.g., K. lactis [Louvencourt et al.,J. Bacteriol., 737 (1983)], K. fragilis, K. bulgaricus, K.thermotolerans, and K. marxianus, yarrowia [EP 402,226], Pichia pastoris[EP 183,070; Sreekrishna et al., J. Basic Microbiol., 28:265-278(1988)], Candida, Trichoderma reesia [EP 244,234], Neurospora crassa[Case et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263 (1979)], andfilamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium[WO 91/00357 published Jan. 10, 1991], and Aspergillus hosts such as A.nidulans [Ballance et al., Biochem. Biophys. Res. Commun., 112: 284-289(1983); Tilbum, et al., Gene, 26:205-221 (1983); Yelton et al., Proc.Natl. Acad. Sci. USA, 81:1470-1474 (1984)] and A. niger [Kelly andHynes, EMBO J., 4: 475-479(1985)].

Suitable host cells for the expression of glycosylated mpl ligand arederived from multicellular organisms. Such host cells are capable ofcomplex processing and glycosylation activities. In principle, anyhigher eukaryotic cell culture is workable, whether from vertebrate orinvertebrate culture. Examples of invertebrate cells include plant andinsect cells. Numerous baculoviral strains and variants andcorresponding permissive insect host cells from hosts such as Spodopterafrugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus(mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori havebeen identified. See, e.g., Luckow et al., Bio/Technology, 6:47-55(1988); Miller et al., in Genetic. Engineering, Setlow, J. K. et al.,eds., Vol. 8 (Plenum Publishing, 1986), pp. 277-279; and Maeda et al.,Nature, 315: 592-594 (1985). A variety of viral strains for transfectionare publicly available, e.g., the L-1 variant of Autographa californicaNPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be usedas the virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,and tobacco can be utilized as hosts. Typically, plant cells aretransfected by incubation with certain strains of the bacteriumAgrobacterium tumefaciens, which has been previously manipulated tocontain the mpl ligand DNA. During incubation of the plant cell culturewith A. tumefaciens, the DNA encoding the mpl ligand is transferred tothe plant cell host such that it is transfected, and will, underappropriate conditions, express the mpl ligand DNA. In addition,regulatory and signal sequences compatible with plant cells areavailable, such as the nopaline synthase promoter and polyadenylationsignal sequences. Depicker et al., J. Mol. Appl. Gen., 1:561 (1982). Inaddition, DNA segments isolated from the upstream region of the T-DNA780 gene are capable of activating or increasing transcription levels ofplant-expressible genes in recombinant DNA-containing plant tissue. EP321,196 published Jun. 21, 1989.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure in recent years [Tissue Culture, Academic Press, Kruse andPatterson, editors (1973)]. Examples of useful mammalian host cell linesare monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651);human embryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al. J. Gen Virol., 36:59 [1977]); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216[1980]); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251[1980];); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.,383: 44-68 [1982]); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2).

Host cells are transfected and preferably transformed with theabove-described expression or cloning vectors of this invention andcultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transfection refers to the taking up of an expression vector by a hostcell whether or not any coding sequences are in fact expressed. Numerousmethods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄ and electroporation. Successful transfection is generallyrecognized when any indication of the operation of this vector occurswithin the host cell.

Transformation means introducing DNA into an organism so that the DNA isreplicable, either as an extrachromosomal element or by chromosomalintegrant. Depending on the host cell used, transformation is done usingstandard techniques appropriate to such cells. The calcium treatmentemploying calcium chloride, as described in section 1.82 of Sambrook etal., supra, is generally used for prokaryotes or other cells thatcontain substantial cell-wall barriers. Infection with Agrobacteriumtumefaciens is used for transformation of certain plant cells, asdescribed by Shaw et al., Gene, 23: 315 (1983) and WO 89/05859 publishedJun. 29, 1989. In addition, plants may be transfected using ultrasoundtreatment as described in WO 91/00358 published Jan. 10, 1991. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456-457(1978) is preferred. General aspects of mammalian cell host systemtransformations have been described by Axel in U.S. Pat. No. 4,399,216issued Aug. 16, 1983. Transformations into yeast are typically carriedout according to the method of Van Solingen et al., J. Bact., 130:946(1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979).However, other methods for introducing DNA into cells such as by nuclearinjection, electroporation, or protoplast fusion may also be used.

E. Culturing the Host Cells

Prokaryotic cells used to produce the mpl ligand polypeptide of thisinvention are cultured in suitable media as described generally inSambrook et al., supra.

The mammalian host cells used to produce the mpl ligand of thisinvention may be cultured in a variety of media. Commercially availablemedia such as Ham's F10 (Sigma), Minimal Essential Medium ([MEM],Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium([DMEM], Sigma) are suitable for culturing the host cells. In addition,any of the media described in Ham and Wallace, Meth. Enz., 58: 44(1979), Barnes and Sato, Anal. Biochem., 102:255 (1980), U.S. Pat. Nos.4,767,704; 4,657,866; 4,927,762; or U.S. Pat. No. 4,560,655; WO90/03430; WO 87/00195; U.S. Pat. Re. No. 30 30,985; or copending U.S.Ser. No. 07/592,107 or Ser. No. 07/592,141, both filed in Oct. 3, 1990,the disclosures of all of which are incorporated herein by reference,may be used as culture media for the host cells. Any of these media maybe supplemented as necessary with hormones and/or other growth factors(such as insulin, transferrin, or epidermal growth factor), salts (suchas sodium chloride, calcium, magnesium, and phosphate), buffers (such asHEPES), nucleosides (such as adenosine and thymidine), antibiotics (suchas Gentamycin™ drug), trace elements (defined as inorganic compoundsusually present at final concentrations in the micromolar range), andglucose or an equivalent energy source. Any other necessary supplementsmay also be included at appropriate concentrations that would be knownto those skilled in the art. The culture conditions, such astemperature, pH, and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

The host cells referred to in this disclosure encompass cells in invitro culture as well as cells that are within a host animal.

F. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, northernblotting to quantitate the transcription of mRNA (Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 (1980]), dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Various labels may be employed, most commonlyradioisotopes, particularly ³²P. However, other techniques may also beemployed, such as using biotin-modified nucleotides for introductioninto a polynucleotide. The biotin then serves as the site for binding toavidin or antibodies, which may be labeled with a wide variety oflabels, such as radionuclides, fluorescers, enzymes, or the like.Alternatively, antibodies may be employed that can recognize specificduplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybridduplexes or DNA-protein duplexes. The antibodies in turn may be labeledand the assay may be carried out where the duplex is bound to a surface,so that upon the formation of duplex on the surface, the presence ofantibody bound to the duplex can be detected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of tissue sections andassay of cell culture or body fluids, to quantitate directly theexpression of gene product. With immunohistochemical stainingtechniques, a cell sample is prepared, typically by dehydration andfixation, followed by reaction with labeled antibodies specific for thegene product coupled, where the labels are usually visually detectable,such as enzymatic labels, fluorescent labels, luminescent labels, andthe like. A particularly sensitive staining technique suitable for usein the present invention is described by Hsu et al., Am. J. Clin. Path.,75:734-738 (1980).

Antibodies useful for immunohistochemical staining and/or assay ofsample fluids may be either monoclonal or polyclonal, and may beprepared in any mammal. Conveniently, the antibodies may be preparedagainst a native mpl ligand polypeptide or against a synthetic peptidebased on the DNA sequences provided herein as described further below.

G. Purification of mpl Ligand Polypeptide

Mpl ligand preferably is recovered from the culture medium as a secretedpolypeptide, although it also may be recovered from host cell lysateswhen directly expressed without a secretory signal.

When mpl ligand is expressed in a recombinant cell other than one ofhuman origin, the mpl ligand is completely free of proteins orpolypeptides of human origin. However, it is necessary to purify mplligand from recombinant cell proteins or polypeptides to obtainpreparations that are substantially homogeneous as to mpl ligand. As afirst step, the culture medium or lysate is centrifuged to removeparticulate cell debris. The membrane and soluble protein fractions arethen separated. The mpl ligand may then be purified from the solubleprotein fraction and from the membrane fraction of the culture lysate,depending on whether the mpl ligand is membrane bound. Mpl ligandthereafter is purified from contaminant soluble proteins andpolypeptides, with the following procedures being exemplary of suitablepurification procedures: by fractionation on immunoaffinity orion-exchange columns; ethanol precipitation; reverse phase HPLC;chromatography on silica or on a cation-exchange resin such as DEAE;chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gelfiltration using, for example, Sephadex G-75; ligand affinitychromatography, using, e.g., mpl and protein A Sepharose columns toremove contaminants such as IgG.

mpl ligand variants in which residues have been deleted, inserted, orsubstituted are recovered in the same fashion as native mpl ligand,taking account of any substantial changes in properties occasioned bythe variation. For example, preparation of a mpl ligand fusion withanother protein or polypeptide, e.g., a bacterial or viral antigen,facilitates purification; an immunoaffinity column containing antibodyto the antigen can be used to adsorb the fusion polypeptide.Immunoaffinity columns such as a rabbit polyclonal anti-mpl ligandcolumn can be employed to absorb the mpl ligand variant by binding it toat least one remaining immune epitope. Alternatively, the mpl ligand maybe purified by affinity chromatography using a purified mpl-IgG coupledto a (preferably) immobilized resin such as Affi-Gel 10 (Bio-Rad,Richmond, Calif.) or the like, by means well known in the art. Aprotease inhibitor such as phenyl methyl sulfonyl fluoride (PMSF) alsomay be useful to inhibit proteolytic degradation during purification,and antibiotics may be included to prevent the growth of adventitiouscontaminants. One skilled in the art will appreciate that purificationmethods suitable for native mpl ligand may require modification toaccount for changes in the character of mpl ligand or its variants uponexpression in recombinant cell culture.

H. Covalent Modifications of mpl Ligand Polypeptide

Covalent modifications of mpl ligand polypeptides are included withinthe scope of this invention. Both native mpl ligand and amino acidsequence variants of the mpl ligand may be covalently modified. One typeof covalent modification included within the scope of this invention isa mpl ligand fragment. Variant mpl ligand fragments having up to about40 amino acid residues may be conveniently prepared by chemicalsynthesis or by enzymatic or chemical cleavage of the full-length orvariant mpl ligand polypeptide. Other types of covalent modifications ofthe mpl ligand or fragments thereof are introduced into the molecule byreacting targeted amino acid residues of the mpl ligand or fragmentsthereof with an organic derivatizing agent that is capable of reactingwith selected side chains or the N- or C-terminal residues.

Cysteinyl residues most commonly are reacted with a-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,a-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Parabromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1M sodium cacodylate at pH 6.Q.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing a-amino-containing residues includeimidbesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chlorobdrohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵I or ¹³¹I to prepare labeled proteinsfor use in radioimmunoassay, the chloramine T method described abovebeing suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R—N═C═N—R′), where R and R′ are differentalkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimideor 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore,aspartyl and glutamyl residues are converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Derivatization with bifunctional agents is useful for crosslinking mplligand to a water-insoluble support matrix or surface for use in themethod for purifying anti-mpl ligand antibodies, and vice-versa.Commonly used crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively. Theseresidues are deamidated under neutral or basic conditions. Thedeamidated form of these residues falls within the scope of thisinvention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the a-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, pp. 79-86 [1983]),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

Another type of covalent modification of the mpl ligand polypeptideincluded within the scope of this invention comprises altering thenative glycosylation pattern of the polypeptide. By altering is meantdeleting one or more carbohydrate moieties found in native mpl ligand,and/or adding one or more glycosylation sites that are not present inthe native mpl ligand.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the mpl ligand polypeptide isconveniently accomplished by altering the amino acid sequence such thatit contains one or more of the above-described tripeptide sequences (forN-linked glycosylation sites). The alteration may also be made by theaddition of, or substitution by, one or more serine or threonineresidues to the native mpl ligand sequence (for O-linked glycosylationsites). For ease, the mpl ligand amino acid sequence is preferablyaltered through changes at the DNA level, particularly by mutating theDNA encoding the mpl ligand polypeptide at preselected bases such thatcodons are generated that will translate, into the desired amino acids.The DNA mutation(s) may be made using methods described above under theheading of “Amino Acid Sequence Variants of mpl ligand.”

Another means of increasing the number of carbohydrate moieties on thempl ligand is by chemical or enzymatic coupling of glycosides to thepolypeptide. These procedures are advantageous in that they do notrequire production of the polypeptide in a host cell that hasglycosylation capabilities for N- or O-linked glycosylation. Dependingon the coupling mode used, the sugar(s) may be attached to (a) arginineand histidine, (b) free carboxyl groups, (c) free sulfhydryl groups suchas those of cysteine, (d) free hydroxyl groups such as those of serine,threonine, or hydroxyproline, (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan, or (f) the amide group ofglutamine. These methods are described in WO 87/05330 published Sep. 11,1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306(1981).

Removal of carbohydrate moieties present on the mpl ligand polypeptidemay be accomplished chemically or enzymatically. Chemicaldeglycosylation requires exposure of the polypeptide to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving thepolypeptide intact. Chemical deglycosylation is described by Hakimuddin,et al., Arch. Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal.Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., Meth. Enzymol.,138:350 (1987).

Glycosylation at potential glycosylation sites may be prevented by theuse of the compound tunicamycin as described by Duskin et al., J. Biol.Chem., 257:3105 (1982). Tunicamycin blocks the formation ofprotein-N-glycoside linkages.

Another type of covalent modification of mpl ligand comprises linkingthe mpl ligand polypeptide to one of a variety of nonproteinaceouspolymers, e.g., polyethylene glycol, polypropylene glycol, orpolyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or U.S. Pat. No. 4,179,337.

It will be appreciated that some screening of the recovered mpl ligandvariant will be needed to select the optimal variant for binding to ampl and having the immunological and/or biological activity definedabove. One can screen for stability in recombinant cell culture or inplasma (e.g., against proteolytic cleavage), high affinity to a mplmember, oxidative stability, ability to be secreted in elevated yields,and the like. For example, a change in the immunological character ofthe mpl ligand polypeptide, such as affinity for a given antibody, ismeasured by a competitive-type immunoassay. Other potentialmodifications of protein or polypeptide properties such as redox orthermal stability, hydrophobicity, or susceptibility to proteolyticdegradation are assayed by methods well known in the art.

4. Preparation of Antibodies to the mpl Ligand

Polyclonal antibodies to the mpl ligand polypeptide generally are raisedin animals by multiple subcutaneous (sc) or intraperitoneal (ip)injections of the mpl ligand polypeptide and an adjuvant. It may beuseful to conjugate the mpl ligand polypeptide (including fragmentscontaining a specific amino acid sequence) to a protein that isimmunogenic in the species to be immunized, e.g., keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsininhibitor using a bifunctional or derivatizing agent, for example,maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteineresidues), N-hydroxysuccinimide (through lysine residues),glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, where R and R¹are different alkyl groups.

The route and schedule of immunizing a host animal or removing andculturing antibody-producing cells are generally in keeping withestablished and conventional techniques for antibody stimulation andproduction. While mice are frequently employed as the host animal, it iscontemplated that any mammalian subject including human subjects orantibody-producing cells obtained therefrom can be manipulated accordingto the processes of this invention to serve as the basis for productionof mammalian, including human, hybrid cell lines.

Animals are typically immunized against the immunogenic conjugates orderivatives by combining 1 mg or 1 μg of mpl ligand conjugate (forrabbits or mice, respectively) with 3 volumes of Freund's completeadjuvant and injecting the solution intradermally at multiple sites. Onemonth later the animals are boosted with ⅕ to {fraction (1/10)} theoriginal amount of conjugate in Freund's complete adjuvant (or othersuitable adjuvant) by subcutaneous injection at multiple sites. Seven to14 days later animals are bled and the serum is assayed for anti-mplligand polypeptide titer. Animals are boosted until the titer plateaus.Preferably, the animal is boosted with the conjugate of the same mplligand polypeptide, but conjugated to a different protein and/or througha different cross-linking agent. Conjugates also can be made inrecombinant cell culture as protein fusions. Also, aggregating agentssuch as alum are used to enhance the immune response.

Monoclonal antibodies are prepared by recovering immune cells—typicallyspleen cells or lymphocytes from lymph node tissue—from immunizedanimals and immortalizing the cells in conventional fashion, e.g., byfusion with myeloma cells or by Epstein-Barr (EB)-virus transformationand screening for clones expressing the desired antibody. The hybridomatechnique described originally by Kohler and Milstein, Eur. J. Immunol.,6:511 (1976) and also described by Hammerling et al., In: MonoclonalAntibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981) hasbeen widely applied to produce hybrid cell lines that secrete highlevels of monoclonal antibodies against many specific antigens.

It is possible to fuse cells of one species with another. However, it ispreferable that the source of the immunized antibody-producing cells andthe myeloma be from the same species.

The hybrid cell lines can be maintained in culture in vitro in cellculture media. The cell lines of this invention can be selected and/ormaintained in a composition comprising the continuous cell line inhypoxanthine-aminopterin thymidine (HAT) medium. In fact, once thehybridoma cell line is established, it can be maintained on a variety ofnutritionally adequate media. Moreover, the hybrid cell lines can bestored and preserved in any number of conventional ways, includingfreezing and storage under liquid nitrogen. Frozen cell lines can berevived and cultured indefinitely with resumed synthesis and secretionof monoclonal antibody.

The secreted antibody is recovered from tissue culture supernatant byconventional methods such as precipitation, ion exchange chromatography,affinity chromatography, or the like. The antibodies described hereinare also recovered from hybridoma cell cultures by conventional methodsfor purification of IgG or IgM, as the case may be, that heretofore havebeen used to purify these immunoglobulins from pooled plasma, e.g.,ethanol or polyethylene glycol precipitation procedures. The purifiedantibodies are sterile filtered, and optionally are conjugated to adetectable marker such as an enzyme or spin label for use in diagnosticassays of the mpl ligand in test samples.

While routinely mouse monoclonal antibodies are used, the invention isnot so limited; in fact, human antibodies may be used and may prove tobe preferable. Such antibodies can be obtained by using human hybridomas(Cote et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 [1985]). In fact, according to the, invention, techniques developedfor the production of chimeric antibodies (Morrison et al., Proc. Natl.Acad. Sci., 81:6851 [1984]; Neuberger et al., Nature, 312:604 [1984];Takeda et al., Nature, 314:452 [1985]; EP 184,187; EP 171,496; EP173,494; PCT WO 86/01533; Shaw et al., J. Nat. Canc. Inst., 80:1553-1559[1988]; Morrison, Science, 229:1202-1207 [1985]; and Oi et al., BioTechniques, 4:214 [1986]) by splicing the genes from a mouse antibodymolecule of appropriate antigen specificity together with genes from ahuman antibody molecule of appropriate biological activity (such asability to activate human complement and mediate ADCC) can be used; suchantibodies are within the scope of this invention.

Techniques for creating recombinant DNA versions of the antigen-bindingregions of antibody molecules (known as Fab fragments), which bypass thegeneration of monoclonal antibodies, are encompassed within the practiceof this invention. One extracts antibody-specific messenger RNAmolecules from immune system cells taken from an immunized animal,transcribes these into complementary DNA (cDNA), and clones the cDNAinto a bacterial expression system. One example of such a techniquesuitable for the practice of this invention was developed by researchersat Scripps/Stratagene, and incorporates a proprietary bacteriophagelambda vector system that contains a leader sequence that causes theexpressed Fab protein to migrate to the periplasmic space (between thebacterial cell membrane and the cell wall) or to be secreted. One canrapidly generate and screen great numbers of functional Fab fragmentsfor those that bind the antigen. Such mpl ligand-binding molecules (Fabfragments with specificity for the mpl ligand polypeptide) arespecifically encompassed within the term “antibody” as it is defined,discussed, and claimed herein.

IV. Therapeutic Use of the Megakaryocytopoietic Protein

The biologically active mpl ligand having hematopoetic effector functionand referred to here as a megakaryocytopoietic protein may be used in asterile pharmaceutical preparation or formulation to stimulatethrombopoeitic activity in patients suffering from thrombocytopenia dueto impaired production, sequestration, or increased destruction ofplatelets. Thombocytopenias associated bone marrow hypoplasia (e.g.,aplastic anemia following chemotherapy or bone marrow transplant) may beeffectively treated with the compounds of this invention as well asdisorders such as disseminated intravascular coagulation (DIC), immunethrombocytopenia (including HIV-induced ITP and non HIV-induced ITP) andthrombotic thrombocytopenia. Additionally, these megakaryocyteopoeiticproteins may be useful in treating meyloproliferative thrombocytoticdiseases as well as thrombocytosis from inflammatory conditions and iniron deficiency.

Still other disorders usefully treated with the megakaryocyteopoeiticproteins of this invention include defects or damage to plateletsresulting from drugs, poisoning or activation on artificial surfaces. Inthese cases, the instant compounds may be employed to stimulate“shedding” of, new “undamaged” platelets. For a more complete list ofuseful applications, see the “Background” supra, especially section(a)-(f) and references cited therein.

The megakaryocyteopoeitic proteins of the instant invention may beemployed alone or in combination with other cytokines, hematopoietins,interleukins, growth factors, or antibodies in the treatment of theabove-identified disorders and conditions. Thus, the instant compoundsmay be employed in combination with other protein or peptide havingthrombopoeitic activity including; G-CSF, CSF-1, GM-CSF, M-CSF, IL-1,IL-3, IL-4, IL-5, erythropoietin (EPO), IL-6, IL-7, and IL-8.

The megakaryocyteopoeitic proteins of the instant invention are preparedin a mixture with a pharmaceutically acceptable carrier. Thistherapeutic composition can be administered orally, intravenously orthrough the nose or lung. The composition may also be administeredparenterally or subcutaneously as desired. When administeredsystematically, the therapeutic composition should be pyrogen-free andin a parenterally acceptable solution having due regard for pH,isotonicity, and stability. These conditions are known to those skilledin the art. Briefly, dosage formulations of the compounds of the presentinvention are prepared for storage or administration by mixing thecompound having the desired degree of purity with physiologicallyacceptable carriers, excipients, or stabilizers. Such materials arenon-toxic to the recipients at the dosages and concentrations employed,and include buffers such as phosphate, citrate, acetate and otherorganic acid salts; antioxidants such as ascorbic acid; low molecularweight (less than about ten residues) peptides such as polyarginine,proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidinone; amino acids such asglycine, glutamic acid, aspartic acid, or arginine; monosaccharides,disaccharides, and other carbohydrates including cellulose or itsderivatives, glucose, mannose, or dextrins; chelating agents such asEDTA; sugar alcohols such as mannitol or sobitol; counterions such assodium and/or nonionic surfactants such as Tween, Pluronics orpolyethyleneglycol.

About 0.5 to 500 mg of a compound or mixture of themegakaryocyteopoeitic protein as the free acid or base form or as apharmaceutically acceptable salt, is compounded with a physiologicallyacceptable vehicle, carrier, excipient, binder, preservative,stabilizer, flavor, etc., as called for by accepted pharmaceuticalpractice. The amount of active ingredient in these compositions is suchthat a suitable dosage in the range indicated is obtained.

Typical adjuvants which may be incorporated into tablets, capsules andthe like are a binder such as acacia, corn starch or gelatin; anexcipient such as microcrystalline cellulose; a disintegrating agentlike corn starch or alginic acid; a lubricant such as magnesiumstearate; a sweetening agent such as sucrose or lactose; a flavoringagent such as peppermint, wintergreen or cherry. When the dosage form isa capsule, in addition to the above materials it may also contain aliquid carrier such as a fatty oil. Other materials of various types maybe used as coatings or as modifiers of the physical form of the dosageunit. A syrup or elixer may contain the active compound, a sweetenersuch as sucrose, preservatives like propyl paraben, a coloring agent anda flavoring agent such as cherry. Sterile compositions for injection canbe formulated according to conventional pharmaceutical practice. Forexample, dissolution or suspension of the active compound in a vehiclesuch as water or naturally occurring vegetable oil like sesame, peanut,or cottonseed oil or a synthetic fatty vehicle like ethyl oleate or thelike may be desired. Buffers, preservatives, antioxidants and the likecan be incorporated according to accepted pharmaceutical practice.

Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing thepolypeptide, which matrices are in the form of shaped articles, e.g.,films, or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels [e.g., poly(2-hydroxyethyl-methacrylate) asdescribed by Langer et al., J. Biomed. Mater. Res., 15:167-277 [1981]and Langer, Chem. Tech., 12:98-105 [1982] or poly(vinylalcohol)],polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers,22:547-556 [1983]), non-degradable ethylene-vinyl acetate (Langer etal., supra), degradable lactic acid-glycolic acid copolymers such as theLupron Depot™ (injectable microspheres composed of lactic acid-glycolicacid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyricacid (EP 133,988).

While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated proteinsremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for protein stabilization depending on themechanism involved. For example, if the aggregation mechanism isdiscovered to be intermolecular S—S bond formation throughthio-disulfide interchange, stabilization may be achieved by modifyingsulfhydryl residues, lyophilizing from acidic solutions, controllingmoisture content, using appropriate additives, and developing specificpolymer matrix compositions.

Sustained-release megakaryocyteopoeitic protein compositions alsoinclude liposomally entrapped megakaryocyteopoeitic protein. Liposomescontaining megakaryocyteopoeitic protein are prepared by methods knownper se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA,82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA,77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP142,641; Japanese patent application 83-118008; U.S. Pat. Nos. 4,485,045and 4,544,545; and EP 102,324. Ordinarily the lipbsomes are of the small(about 200-800 Angstroms) unilamellar type in which the lipid content isgreater than about 30 mol. % cholesterol, the selected proportion beingadjusted for the optimal megaokarycyteopoeitic protein therapy.

The dosage will be determined by the attending physician taking intoconsideration various factors known to modify the action of drugsincluding severity and type of disease, body weight, sex, diet, time androute of administration, other medications and other relevant clinicalfactors. Typically, the daily regimen will range from 1-3000 μg/kg bodyweight. Preferably the dosage will range from 10-1000 μg/kg body weight.Most preferably, the dosage will range from 50 to 150 mg/day.Therapeutically effective dosages may be determined by either in vitroor in vivo methods.

EXAMPLES

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and illustrativeexamples, make and utilize the present invention to the fullest extent.The following working examples therefore specifically point outpreferred embodiments of the present invention, and are not to beconstrued as limiting in any way of the remainder of the disclosure.

Example I

Partial Purification of the MPL-ligand

Aplastic porcine plasma obtained from irradiated pigs is made 4M withNaCl and stirred for 30 min. at room temperature. The resultantprecipitate is removed by centrifugation at 3800 rpm in a Sorvall RC3Band the supernatant is loaded onto a Phenyl-Toyopearl columnequilibrated in 10 mM NaPO₄ containing 4M NaCl. The column is washedwith this buffer until A₂₈₀ is <0.05 and eluted with dH₂O. The elutedprotein peak is diluted with dH₂O to a conductivity of 15 mS and loadedonto a Blue-Sepharose column equilibrated in PBS. Subsequently, thecolumn is washed with 5 column volumes each of PBS and 10 mM NaPO₄ (pH7.4) containing 2M urea. Proteins are eluted from the column with 10 mMNaPO₄ (pH 7.4) containing 2M urea and 1M NaCl. The eluted protein peakis made 0.01% octyl glucoside(n-octyl β-D-glucopyranoside) and 1 mM eachwith EDTA and Pefabloc and loaded directly onto tandemly linked CD4-IgGand mpl-IgG ultralink (Pierce) columns (see below). The CD4-IgG columnis removed after the sample is loaded and the mpl-IgG column is washedwith 10 column volumes each of PBS and PBS containing 2 M NaCl andeluted with 0.1M glycine-HCL pH 2.25. Fractions are collected into{fraction (1/10)}th volume 1M tris pH 8.0.

Analysis of eluted fractions from the mpl-affinity column by SDS-PAGE(4-20%, novex gel) run under reducing conditions, reveal the presence ofseveral proteins (FIG. 3 ). Proteins that silver stain with thestrongest intensity resolve with apparent Mr of 66,000, 55,000, 30,000,28,000 and 14,000. To determine which of these proteins stimulateproliferation Ba/F3-mpl cell cultures these proteins were eluted fromthe gel as described in a Example II below.

Ultralink Affinity Columns

1.0-20 mg of mpl-IgG or CD4-IgG in PBS are coupled to 0.5 grams ofUltralink resin (Pierce) as described by the manufactures instructions.

Construction and Expression of mpl-IgG

A chimeric molecule comprising the entire extracelluar domain of humanmpl P (amino acids 1-491) and the Fc region of a human IgG1 molecule wasexpressed in 293 cells. A cDNA fragment corresponding to amino acids1-491 of human mpl P was obtained by PCR from a human platelet cDNAlibrary. A ClaI site was inserted at the 5′ end and a BstEII site at the3′ end. The fragment was cloned upstream of the IgGI Fc region in aBluescript vector between the ClaI and. the BstEII sites after partialdigestion of the PCR product with BstEII because of 2 other BstEII sitespresent in the extracellular domain of mpl. The BstEII site introducedat the 3′ end of the mpl PCR product was designed to have the Fc regionin frame with the mpl extracellular domain. The construct was subclonedinto pRK5-tkneo vector between the ClaI and Xbal sites and transfectedinto 293 human embryonic kidney cells by the calcium phosphate method.The cells were selected in 0.4 mg/ml G418 and individual clones wereisolated. mpl-IgG expression from isolated clones was determined using ahuman Fc specific ELISA. One clone had the highest expression of 1-2mg/ml.

Example II

Highly Purified mpl Ligand

Gel Elution Protocol

Equal amounts of affinity purified mpl ligand (fraction 6 eluted fromthe mpl-IgG column) and 2× Laemmli sample buffer were mixed at roomtemperature without reducing agent and loaded onto a Novex 4-20%polyacrylamide gel as quickly as possible. The sample was not heated. Asa control, sample buffer without ligand was run in an adjacent lane. Thegel was run at 4-6 C. at 135 volts for approximately 2¼ hours. Therunning buffer was initially at room temperature. The gel was thenremoved from the gel box and the plate on one side of the gel removed.

A replica of the gel was made on nitrocellulose as follows: A piece ofnitrocellulose was wet with distilled water and carefully laid on top ofthe exposed gel face so air bubbles were excluded. Fiducial marks wereplaced on the nitrocellulose and the gel plate so the replica could beaccurately repositioned after staining. After approximately 2 minutes,the nitrocellulose was carefully removed, and the gel was wrapped inplastic wrap and placed in the refrigerator. The nitrocellulose wasstained with Biorad's gold total protein stain by first agitating it. in3×10 mL 0.1% Tween 20+0.5 M NaCl+0.1 M Tris-HCl pH 7.5 overapproximately 45 minutes followed by 3×10 mL purified water over 5minutes. The gold stain was then added and allowed to develop until thebands in the standards were visible. The replica was then rinsed withwater, placed over the plastic wrap on the gel and carefully alignedwith the fiducial marks. The positions of the Novex standards weremarked on the gel plate and lines were drawn to indicate the cuttingpositions. The nitrocellulose and plastic wrap were then removed and thegel cut along the indicated lines with a sharp razor blade. The cutswere extended beyond the sample lanes so they could be used to determinethe positions of the slices when the gel was stained. After the sliceswere removed, the remaining gel was silver stained and the positions ofthe standards and the cut marks measured. The molecular weightscorresponding to the cut positions were determined from the Novexstandards.

The 12 gel slices were placed into the cells in two Biorad model 422electro-eluters. 12-14K molecular weight cutoff membrane caps were usedin the cells. 50 mM ammonium bicarbonate+0.05% SDS (approximately pH7.8) was the elution buffer. 1 L of buffer was chilled approximately 1hour in a 4-6 C. coldroom before use. Gel slices were eluted at 10ma/cell (40 v initially) in a 4-6 C. coldroom. Elution tookapproximately 4 hours. The cells were then carefully removed and theliquid above the frit removed with a pipet. The elution chamber wasremoved and any liquid above the membrane cap removed with a pipet. Theliquid in the membrane cap was removed with a Pipetman and saved. 50 uLaliquots of purified water were then placed in the cap, agitated andremoved until all the SDS crystals dissolved. These washes were combinedwith the saved liquid above. Total elution sample volume was 300-500 uLper gel slice. Samples were placed in 10 mm Spectrapor 4 12-14K cutoffdialysis tubing which had been soaked several hours in purified water.They were dialyzed overnight at 4-6 C. against 600 mL of phosphatebuffered saline (PBS is approximately 4 mM in potassium) per 6 samples.The buffer was replaced the next morning and dialysis continued for 2.5hours. Samples were then removed from the dialysis bags and placed inmicrofuge tubes. The tubes were placed on ice for 1 hour, microfuged at14K rpm for 3 min. and the supernantants carefully removed from theprecipitated SDS. The supernatants were then placed on ice forapproximately 1 hour more and microfuged again for 4 min. Thesupernatants were diluted in phosphate buffered saline and submitted forthe activity assay. Remaining samples were frozen at −70 C.

Example III

Mpl Ligand Microsequencing

Fraction 6 (2.6 ml) from the MPL-IgG affinity column was concentrated ona Microcon-10 (Amicon). In order to prevent the MPL ligand fromabsorbing to the Microcon, the membrane was rinsed with 1% SDS and 5 μlof 10% SDS was added to fraction 6. Sample buffer (20 μl) of 2× wasadded to the fraction #6 after Microcon concentration (20 μl) and thetotal volume (40 μl) was loaded on a single lane of a 4-20% gradientacrylamide gel (Novex). The gel was run following Novex protocol. Thegel was then equilibrated for 5 min. prior to electroblotting in 10 mM3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) buffer, pH 11.0,containing 10% methanol. Electroblotting onto Immobilon-PSQ membranes(Millipore) was carried out for 45 min. at 250 mA constant current in aBioRad Trans-Blot transfer cell (32). The PVDF membrane was stained with0.1% Coomassie Blue R-250 in 40% methanol, 0.1% acetic acid for 1 min.and destained for 2-3 min. with 10% acetic acid in 50% methanol. Theonly proteins that were visable in the Mr 18,000-35,000 region of theblot had Mr of 30,000, 28,000 and 22,000.

Bands at 30, 28 and 22 KDa were subjected to protein sequencing.Automated protein sequencing was performed on a model 470A AppliedBiosystem sequencer equipped with an on-line PTH analyzer. The sequencerwas modified to inject 80-90% of the sample (Rodriguez, H. J.Chromatogr. 350:217-225 [1985]). Acetone (˜12 μl/L) was added to solventA to balance the UV absorbance. EIectroblotted proteins were sequencedin the Blott cartridge. Peaks were integrated with Justice Innovationsoftware using Nelson Analytical 970 interfaces. Sequence interpretationwas performed on a VAX 5900 (Henzel, W. J., Rodriguez, H., and Watanabe,C. J. Chromatogr. 404:41-52 [i987]). N-terminal sequence using oneletter code with uncertain residues in parenthesis of indicated gelbands were:

1) 30 KDa (1.8 pmol)  1       5         10        15         20             25 (S) P A P PA(C)D P R L L N K L L R D D (H/S) V L H (G) R L (SEQ ID NO.:3) 2) 28 KDa(O.5 pmol)  1        5         10        15        20            25 (S)P A P P A X D P R L L N K L L R D D (H) V L (H) G R (SEQ ID NO.:4) 3) 22KDa (O.5 pmol) 1       5         10 X P A P P A X D P R L X (N) (K) (SEQID NO.:5)

Example IV

Liquid Suspension Megakaryocytopoiesis Assay

Human peripheral stem cells (PSC) (obtained from consenting patients)were diluted 5 fold with IMDM media (Gibco) and centrifuged for 15 min.at room temp. at 800×g. The cell pellets were resuspended in IMDM andlayered onto 60% percoll (density 1.077 gm/ml ) (Pharmacia) andcentrifuged at 800×g for 30 min. The light density mononuclear cellswere aspirated at the interface and washed 2× with IMDM and plated outat 1-2×10⁶ cells/ml in IMDM containing 30% FBS (1 ml final volume) in 24well tissue culture clusters (Costar). APP or mpl-ligand depleted APPwas added to 10% and cultures were grown for 12-14 days in a humidifiedincubator at 37 C. in 5% CO₂ and air, The cultures were also grown inthe presence of 10% APP with 0.5 μg of mpl-IgG added at days 0, 2 and 4.APP was depleted of mpl-ligand by passing APP through a mpl-IgG affinitycolumn.

To quanitate megakaryocytopoiesis in these liquid suspension cultures, amodification of (Solberg, L. A. et al. ) was used and employs aradiolabled murine IgG monoclonal antibody (HP1-1D) to GPIIbIIIa(provided by Dr. Nichols-, Mayo Clinic)-. 100 μg of HP1-1D wasradiolabled with 1mCi of Na¹²⁵I using enzymobeads (Biorad, RichmondCalif.) as described by the manufactures instructions. RadiolabledHP1-1D was stored at −70° C. in PBS containing 0.01% octyl-glucoside.Typical specific activities were 1-2×10⁶ cpm/μg (>95% precipital by12.5% trichloroacetic acid).

Liquid suspension cultures were set up in triplicate for eachexperimental point. After 12-14 days in culture the 1 ml cultures weretransferred to 1.5 ml eppendorf tubes and centrifuged at 800×g for 10min. at room temp. and the resultant cell pellets were resuspended in100 μl of PBS containing 0.02% EDTA and 20% bovine calf serum. 10 ng of¹²⁵I-HP1-1D in 50 μl of assay buffer was added to the resuspendedcultures and incubated for 60 min. at RT with occasional shaking.Subsequently, cells were collected by centrifugation at 800×g for 10 minat RT and washed 2× with assay buffer. The pellets were counted for 1min. in a gamma counter (Packard). Non-specific binding was determinedby adding 1 ug of unlabled HP1-1D for 60 min. before the addition oflabeled HP1-1D. Specific binding was determined as the total ¹²⁵I-HP1-1D bound minus that bound in the presence of excess unlabled HP1-1D.

Example V

Oligonucleotide PCR Primers

Based on the amino-terminal amino acid sequence obtained from the 30kDa, 28 kDa and 22 kDa proteins, degenerate oligonucleotides weredesigned for use as polymerase chain reaction (PCR) primers. Two primerpools were synthesized, a positive sense 20 mer pool encoding amino acidresidues 2-8 (mpl.1) and a negative sense 21 mer pool encoding aminoacids 18-24 (mpl. 2).

mpl. 1 5′ CCN GCN CCN CCN GCN TGY GA 3′ (SEQ ID NO.: 6) (2,048-folddegenerate)

mpl. 2 5′ NCC RTG NAR NAC RTG RTC RTC 3′ (SEQ ID NO.: 7) (2,048-folddegenerate)

Porcine genomic DNA, isolated from porcine peripheral blood lymphocytes,was used as a template for PCR. The 50 μl reaction contained: 0.8 μg ofporcine genomic DNA in 10 mM Tris pH 8.3, 50 mM KCl, 3 mM MgCl₂, 100ug/ml BSA, 400 μM dNTPs, 1 μM of each primer pool and 2.5 units of Taqpolymerase. Initial template denaturation was at 94° C. for 8 min.followed by 35 cycles of 45 seconds at 94° C., 1 min. at 55° C. and 1min. at 72° C. The final cycle was allowed to extend for 10 min. at 72°C. PCR products were separated by electrophoresis on a 12%polyacrylamide gel and visualized by staining with ethidium bromide. Ifthe amino-terminal amino acid sequence is encoded by a single exon thenthe correct PCR product is expected to be 69 bp. A DNA fragment of thissize was eluted from the gel and subcloned into pGEMT (Promega).Sequences of three clones are shown below:

(1) gemT3

5′ CCAGCGCCGC CAGCCTGTGA CCCCCGACTC CTAAATAAAC TGCCTCGTGA

3′ GGTCGCGGCG GTCGGACACT GGGGGCTGAG GATTTATTTG ACGGAGCACT

TGACCACGTT CAGCACGGC 3′ (SEQ ID NO.: 8) 69

ACTGGTGCAA GTCGTGCCG 5′

(2) gemT7

5′ CCAGCACCTC CGGCATGTGA CCCCCGACTC CTAAATAAAC TGCTTCGTGA

3′ GGTCGTGGAG GCCGTACACT GGGGGCTGAG GATTTATTTG ACGAAGCACT

CGACCACGTC CATCACGGC 3′ (SEQ ID NO.: 9) 69

GCTGGTGCAG GTAGTGCCG 5′

(3) gemT9

                        P  R  L  L  N  K  L  L  R 5′CCAGCACCGCCGGCATGTGACCCCCGACTCCTAAATAAACTGCTTCGTGACG (SEQ ID NO.: 10) 3′GGTCGTGGCGGCCGTACACTGGGGGCTGAGGATTTATTTGACGAAGCACTGC ATCATGTCTATCACGGT3′ (SEQ ID NO.: 11) TAGTACAGATAGTGCCA 5′

The position of the PCR primers is indicated by the underlined bases.These results verify the N-terminal sequence obtained for amino acids9-17 for the 30 kDa, 28 KDa and 22 kDa proteins and indicated that thissequence is encoded by a single exon of porcine DNA.

Based on these results a single oligonucleotide of 53 nucleotides wassynthesized (from AA position 2 to 19), labeled with ³²P and used toscreen porcine and human genomic DNA libraries in order to isolate theporcine and human mpl ligand genes. This “consensus probe” has thefollowing sequence:

5′ CCA GCA CCG CCG GCA TGT GAC CCC CGA CTC CTA AAT AAA CTG CTT CGT GACGA 3′ (SEQ ID NO.: 12)

Example VI

CMK Assay for Thrombopoletin (TPO) Induction of Platelet AntigenGPII_(b)III_(a) Expression

CMK cells are maintained in RMPI 1640 medium (Sigma) supplemented with10% fetal bovine serum and 10 mM glutamine. In preparation for theassay, the cells are harvested, washed and resuspended at 5×10⁵ cells/mlin serum-free GIF medium supplemented with 5 mg/L bovine insulin, 10mg/L apo-transferrin, 1× trace elements. In a 96-well flat-bottom plate,the TPO standard or experimental samples are added to each well atappropriate dilutions in 100 ul volumes. 100 ul of the CMK cellsuspension is added to each well and the plates are incubated at 37° C.,in a 5% CO₂ incubator for 48 hours. After incubation, the plates arespun at 1000 rpm at 4° C. for five minutes. Supernatants are discardedand 100 ul of the FITC-conjugated GPII_(b)III_(a) monoclonal 2D2antibody is added to each well. Following incubation at 4° C. for 1hour, plates are spun again at 1000 rpm for five minutes. Thesupernatants containing unbound antibody are discarded and 200 ul of0.1% BSA-PBS wash is added to each well. The 0.1% BSA-PBS wash step isrepeated three times. Cells are then analyzed on a FASCAN using standardone parameter analysis measuring relative fluorescence intensity.

Example VII

DAMI Assay for Thrombopoietin (TPO) by Measuring Endomitotic Activity ofDAMI Cells on 96-well Microtiter Plates

DAMI cells are maintained in IMDM+10% horse serum (Gibco) supplementedwith 10 mM glutamine, 10 ng/ml Penicillin G, and 50 ug/ml streptomycin.In preparation for the assay, the cells are harvested, washed, andresuspended at 1×10⁶ cells/ml in IMDM+1% horse serum. In a 96-wellround-bottom plate, 100 ul of the TPO standard or experimental samplesis added to DAMI cell suspension. Cells are then incubated for 48 hoursat 37° in a 5% CO₂ incubator. After incubation, plates are spun in aSorvall 6000B centrifuge at 1000 rpm for five minutes at 4° C.Supernatants are discarded and 200 ul of PBS-0.1% BSA wash step isrepeated. Cells are fixed by the addition of 200 ul ice-cold 70%Ethanol-PBS and resuspended by aspiration. After incubation at 4° C. for15 minutes, the plates are spun at 2000 rpm for five minutes and 150 ulof 1 mg/ml RNAse containing 0.1 mg/ml Propidium Iodide and 0.05%Tween-20 is added to each well. Following a one hour incubation at 37°C. the changes in DNA content are measured by flow cytometry. Polyploidyis measured and quantitated as follows:${{Normalized}\quad {Polyploid}\quad {Ratio}\quad ({NPR})} = \frac{\left( {{\% \quad {Cells}\quad {in}} > {{G2} + {{M/\%}\quad {Cells}\quad {in}}} < {{G2} + M}} \right)\quad {with}\quad {TPO}}{\left( {{\% \quad {Cells}\quad {in}} > {{G2} + {{M/\%}\quad {Cells}\quad {in}}} < {{G2} + M}} \right)\quad {in}\quad {control}}$

Example VIII

Thrombopoietin (TPO)in vivo Assay (Mouse Platelet Rebound Assay)

In vivo Assay for ³⁵S Determination of Platelet Production

C57BL6 mice (obtained from Charles River) are injected intraperitoneally(IP) with 1 ml goat anti-mouse platelet serum (6 amps) on day 1 toproduce thrombocytopenia. On days 5 and 6, mice are given two IPinjections of the factor or PBS as the control. On day 7, thirty μCi ofNa₂ ³⁵SO₄ in 0.1 ml saline are injected intravenously and the percent³⁵S incorporation of the injected dose into circulating platelets ismeasured in blood samples obtained from treated and control mice.Platelet counts and leukocyte counts are made at the same time fromblood obtained from the retro-orbital sinus.

While the invention has necessarily been described in conjunction withpreferred embodiments and specific working examples, one of ordinaryskill, after reading the foregoing specification, will be able to effectvarious changes, substitutions of equivalents, and alterations to thesubject matter set forth herein, without departing from the spirit andscope thereof. Hence, the invention can be practiced in ways other thanthose specifically described herein. It is therefore intended that theprotection granted by Letters Patent hereon be limited only by theappended claims and equivalents thereof.

All references cited herein are hereby expressly incorporated byreference.

                   #              SEQUENCE LIS #TING(1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 12(2) INFORMATION FOR SEQ ID NO:1:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 20 amino  #acids           (B) TYPE: Amino Acid          (D) TOPOLOGY: Linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:Ser Pro Ala Pro Pro Ala Cys Asp Pro Arg L #eu Leu Asn Lys Leu  1               5  #                 10  #                 15Leu Arg Asp Asp His                  20 (2) INFORMATION FOR SEQ ID NO:2:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 25 amino #acids           (B) TYPE: Amino Acid           (D) TOPOLOGY: Linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:Ser Pro Ala Pro Pro Ala Cys Asp Pro Arg L #eu Leu Asn Lys Leu  1               5  #                 10  #                 15Leu Arg Asp Asp His Val Leu His Gly Arg                  20 #                 25 (2) INFORMATION FOR SEQ ID NO:3:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 26 amino #acids           (B) TYPE: Amino Acid           (D) TOPOLOGY: Linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:Ser Pro Ala Pro Pro Ala Cys Asp Pro Arg L #eu Leu Asn Lys Leu  1               5  #                 10  #                 15Leu Arg Asp Asp Xaa Val Leu His Gly Arg L #eu                  20 #                 25  # 26 (2) INFORMATION FOR SEQ ID NO:4:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 25 amino #acids           (B) TYPE: Amino Acid           (D) TOPOLOGY: Linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:Ser Pro Ala Pro Pro Ala Xaa Asp Pro Arg L #eu Leu Asn Lys Leu  1               5  #                 10  #                 15Leu Arg Asp Asp His Val Leu His Gly Arg                  20 #                 25 (2) INFORMATION FOR SEQ ID NO:5:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 14 amino #acids           (B) TYPE: Amino Acid           (D) TOPOLOGY: Linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:Xaa Pro Ala Pro Pro Ala Xaa Asp Pro Arg L #eu Xaa Asn Lys  1               5  #                 10  #             14(2) INFORMATION FOR SEQ ID NO:6:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 20 base  #pairs           (B) TYPE: Nucleic Acid          (C) STRANDEDNESS: Single           (D) TOPOLOGY: Linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:CCNGCNCCNC CNGCNTGYGA             #                  #                   # 20 (2) INFORMATION FOR SEQ ID NO:7:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 21 base  #pairs          (B) TYPE: Nucleic Acid           (C) STRANDEDNESS: Single          (D) TOPOLOGY: Linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:NCCRTGNARN ACRTGRTCRT C            #                  #                   #21 (2) INFORMATION FOR SEQ ID NO:8:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 69 base  #pairs          (B) TYPE: Nucleic Acid           (C) STRANDEDNESS: Double          (D) TOPOLOGY: Linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:CCAGCGCCGC CAGCCTGTGA CCCCCGACTC CTAAATAAAC TGCCTCGTGA  #              50 TGACCACGTT CAGCACGGC             #                  #                   #  69 (2) INFORMATION FOR SEQ ID NO:9:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 69 base  #pairs          (B) TYPE: Nucleic Acid           (C) STRANDEDNESS: Double          (D) TOPOLOGY: Linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:CCAGCACCTC CGGCATGTGA CCCCCGACTC CTAAATAAAC TGCTTCGTGA  #              50 CGACCACGTC CATCACGGC             #                  #                   #  69 (2) INFORMATION FOR SEQ ID NO:10:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 9 amino  #acids          (B) TYPE: Amino Acid           (D) TOPOLOGY: Linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:Pro Arg Leu Leu Asn Lys Leu Leu Arg   1               5 #              9 (2) INFORMATION FOR SEQ ID NO:11:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 69 base  #pairs          (B) TYPE: Nucleic Acid           (C) STRANDEDNESS: Double          (D) TOPOLOGY: Linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:CCAGCACCGC CGGCATGTGA CCCCCGACTC CTAAATAAAC TGCTTCGTGA  #              50 CGATCATGTC TATCACGGT             #                  #                   #  69 (2) INFORMATION FOR SEQ ID NO:12:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 53 base  #pairs          (B) TYPE: Nucleic Acid           (C) STRANDEDNESS: Single          (D) TOPOLOGY: Linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:CCAGCACCGC CGGCATGTGA CCCCCGACTC CTAAATAAAC TGCTTCGTGA  #              50 CGA                  #                  #                   #              53

We claim:
 1. An isolated substantially homogeneous naturally occuringPorcine mpl ligand.
 2. The isolated substantially homogeneous mpl ligandof claim 1 characterized in that: (1) the ligand stimulates theincorporation of labeled nucleotides (³H-thymidine) into the DNA of IL-3dependant Ba/F3 cells transfected with human mpl P; and (2) theamino-terminal sequence is SPAPPACDPRLLNKLLRDDHVLHGR (SEQ ID NO: 2). 3.A composition comprising the mpl ligand of claim 1 and apharmaceutically acceptable carrier.